Note: Descriptions are shown in the official language in which they were submitted.
W 095/09182 ~ 2 PCT/AU94/00573
PROTECTIVE ANTIGENS AGAINST PARASITES
~ A~AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
The present invention relates to ~rllibody ~.robes and the use of such
probes in the dele-,1ioll and p~liricdtioll of a number of prole;li~/e and diagnostic
antigens from parasites and ba~ ,ia. In particular, the present invention relates
s to the ide~ calion and puliric~liGn of a~liye~ls in the parasites Oste,l~ia
circu",c;"~td. Tricho:jl,u"~lus colut,rirul",;s and F~C~ dl;~ plt~JdrdliGI) of
vaccines incol~.Grdli"y the above a"liyel~s and use of such antigens in diagnostic
assays.
Considerable effort has been devoted in the prior art to the dcvelopl),e,ll
of vaccines to control par~sitic, bacterial and other illr~.;liol~s of animals including
liveslock. However, little ,uru~r~ss has been achieved to this end in the past 5years although the ~ssoci~ter~ tecl~no'ogy of producing ron,i~" products in large
amounts in eukaryotic and prokaryotic ~rya~ r"s has advanced e~ol",ously. The
ide"Lir~cdlion of prote.;tive antigens in illlpol1dnl pathogenic ~llre~tiG"s in animals,
for example, has remained the principal stumbling block to the generation of
vaccines.
In Ausll dlian Patent No. 640364, the entire ~lisclosl ~re of which is
incorporated herein by ~ererel,ce, there is described a method for preparing an
antigen ~ssoci~ted with a dise~-se pdllloyell which melllod includes providing asample of a disease pdU,ogen taken from a disease pathogen at a stage of
development during which it is thought to be most susce~Jlible to attack; and anantibody probe including at least one antibody agai"~t a disease pathogen;
probing the pathogen sample with the auliLod~/ probe; and isolating the antigen
detected.
2s Whilst this method provides a significant advance in the art, invesligdlions
were limited to a small number of parasitic and bacterial illre-.1iol Is. It would be a
significant advance in the art if the method could be s~cces~r.llly applied to other
infections and to the idellliricdliGn of further protective dllligells.
- Ostertagia circumcin~.t~ is an inl~sli"al nel"alude parasite of sheep that
localises in the abomasum (4th slo",ach) of sheep. O. circu"~c;". 1;, has recently
been recl~ssified as Te~dorc~i~ circun)cil-ct~ however the latter name is not yet
in common usage. Eggs from adult ,uardsiles in the abol"asum are passed in the
faeces from infected sheep onto the pasture.
WO 95/09182 PCT/AU94/00573
~72~ 2-
After hatchi.~g larval dcvelop~e~l to the third stage (L3) occurs on the
pasture. L3 larvae are ingested by yrd~i~ ,y sheep and undergo further
develG~,",enl in the abor"asum. The U stage dcvelops in the abo",asal crypts;
development may be slowed or ar,esled ~lepe"Jing on the immune status of the
s sheep and on the seaso". Larvae may lie dormant in the mucosal crypts over
winter before dcvelG,ci.lg into mature egg producing adults in the spring. The
suddèn synchronised rise in parasite numbers can cause significant morbidity.
Tissue damage due to feeding of late larval stages and adults on mucosa c~l~ses
serum leakage and hypertrophy of the abGi "asal lining with s' Ihsecluent
lo interference with abomasal function and co"se4uently poor growth of the animal.
A similar pathology is caused in cattle by the related pardsile O. o~
Despite the impGIldl~ce of O~ ia species in causing considerable
ecol,ol"ic losses in the sheep and cattle industry in Australia and overseas no
successful vaccine has been dcveloped in the prior art against this parasite.
Fasciola hepali-:~ (liverfluke) is a parasite belonging to the T~emalode
family which can infect a variety of wild and dGr,leslic animal species and is of
particular economic impGILdnce to the sheep and cattle industry. Among the
species studied rats are the only host that can develop a strong immunologicallybased immunity to reinfection (reviewed in Haroun E T M and G V Hillyer Vet
Parasitol Q 63-93 1986). Antigens strongly recognised by the rat immune
system are therefore of particular importance to vaccination strategies and are
described in the present application. Australian Patent 640364 describes a
protective antigen against Fasciola hepatica infections having an approximate
molecular weight of 120 to 12~ kilodaltons. This antigen was differentially
2s recognised between cattle and sheep and is completely different from the antigens described in the present applicdlion.
Infections with Trichostron~ylus spp. occur in the first 3-4 metres of small
intestine in sheep and cause reduced wool production and body growth illthrift
and scouring. Death can result in severe il,r~;lio"s. It is also an economicallysignificant ~lise~se but no successrul vaccine has been developed in the prior art
against this parasite.
It is accordingly an object of the present invention to overcome or at least
alleviate one or more of the difficulties and deficiencies related to the prior art.
WO 95/09182 2~ 7 2 4 9 2 PCT/AU94/00573
Acco,di"yly in a first aspect the presenl invention provides a putative
prolec;ti./e antigen or rl~y",ent ll,ereof against Ostert~gia circu",~.;"r.1~ or r~lat~d
i"~e~:tio"s selQcted from antigens having a~.~ ruxi,nale mo'~cl~lar wcighls in the
region of 26-36 and 95-105 kilodallùlls as hereinafter described. The a"liyens
may also be ,vn:senl in other species and strains of parasites.
The 26-36 kD 0. circu",~;n.;t~ antigenic region may include a doublet
antigen in the 32-36kD posilio". The doublet antigen may be a lectin-like i3-
g~l~ctosi~le-binding protein. The 32-36 kD ~ouhlet antigen may include one or
more of the peptide sequences
o 1) SAHGPPGQ
2) FPHGPSYQHGYA
3) IVTHPNR
The lower bands in the 26-36 kD 0. circu",~i" ~ antigenic region contain
proteins hor) cl~gous to l,o,l~G",yosin and glul~ll,ior,ille S-t,al):irer~se.
The 0. drculll~jillr-~ sequences hon.Dl~ous to
(A) tropomyosin are:
1. N-terrninal sequence: MKAEEVRQALK
2. Intemal sequence: VEADLERAEERAEMGENKWVL
(B) Glutathionine S-t,dns~r~se:
N-tel"~i"al: VQYKLWFDGRXAAEV
In a further pl~f~lled aspect of the present invention there is provided a
putative protective al ,liyel) or r, ~yl "enl ll ,ereùf agai. ,-~t Tricho~l, on4ylus
colub,ifor",is. or related i"fe~lions having an appr~,~i",dle mol~cul~r weight of 32-
35 kilod~ltons as herci.,dner described. The protecti-/e a"ligell may be a
doublet.
The T. colubrirvl"~;s ai,ligen is in the same position on SDS-PAGE as the
0. circumcincta doublet antigen ,~re"~l to above and it is therefore possible that
these are essentially similar r~ cllles but with species-specific epitopes
recognised most strongly by the hor". '~gous su~ el "dlant antibody probes.
In a still further aspect of the present invention there is provided a
putative protective anliyen or fr~y",enl Iher~or ~gai"sl F~ la he~3lica or
related infections selecte~l from antigens having approxi",dle ",cle~ r weights
in the region of 28 kilodaltons, 32 kilodaltons, 37 kilodaltons 42 to 100
WO 95/09182 ~ 2 4 PCT/AU94/00573
kilodaltons, 54 to ~5 kilodaltons and ~200 kiloddlLur.s, as her~,;..b~:rur~ described.
Similar a,.ligens may be present in other Fasciola species, for example F~-cr~
~i~d,.lica and in other l,~:r"a~ode par~siles for example Schislosû,,,a spp.
The ~200 ki'ocl~ ) F. hP.~ allliyeO may be a doubiet ar,ligen. The
s F. hepdlica antigens are uniquely recogl.ised by super"dlanl from mesenteric
Iymph node (MLN) cells in immune, challenged rats.
The 32 kD al ,ligen may include an N-terminal peptide sequence
KPNYKRQFEPFSDELIHYINLE.
The 54-55 kD antigen may include an N-terminal peptide sequence
LEDNGRTHWAVLVA.
It will be undel:,lood that lecon,bi,.at)l protein antigens may be used in
place of the native ai ligens extracted from the par~siles.
Fray",er,ls of the antigen(s) containing prolecti~e epitopes, and synthetic
peptides containing prote-;ti~e epitopes may be s~ Ihstitl ~ted for the entire native or
recol,lbinant mo'ec~le(s) in both vdcc;"es and diagnostic tests.
The antigen(s) may be pr~se"l in other species of parasites and would
thus be of use in vacc;nalion and diagnosis of other disc:~es besides that caused
by the designaled pathogens.
It will be further underalood that anliLoJy raised agai"st these antigens
may be used in diagnostic tests or as an immunoprophylactic agent, whether
polyclonal or monoclonal.
The protective antigens accGrdi, .9 to the pr~senl invention may be
produced utilising the nl~ll-od as described in Australian Patent 640364, referred
to above. Accordingly, in a further aspect, the present invention provides a
method for preparing an antigen associated with a dise~ce pathogen selected
from Fasciola, Oslt:,la~Jia and Trichosl~u"s~ylus species, and related species, as
described above, which Ill~:Ulod incl~des
providing
a sample of a disease pdlhoy~l) sele.;led from F~ciola. Osterta~ia
and Trichostron~ylus species, and related species; and
a cGr,espol)di"y alllibody probe including at least one antibody
against the respective rlise~se pathogen produced by a method including
WO9~;/09182 ~ 3L s7 2 ~ 9 ~ PCT/AU94/00573
-- 5 --
providing a bial~ic~l sdmple from an immune animal taken a
short time after the immune animal has been challenged with a
,~dll,ogen or pdll,os~en extract selected from F~C~ Osterta~ia
and Tlic~lûallu~ ylus s~ ecies and related species;
s isolating cells from the biological sample;
culturing cells in ~!i~ in a suitable culture medium; and
ha~esting antibodies produced from said cells
probing the ,.)dll,Gyen sdlllple to detect at least one antigen with the
cor,es~.or,cling antibody probe; and
isolating the antigen ~lete~cl.
The disease ~.dtl,ogen sample is preferably a parasite parasite extract or
parasitic section thereof. The Oste,la~ dll,ogen may be OsLt:,lagia
circu",ci"cta or Ostertagi~ osle,~ . The Tricl,o~l,ùn~ylus pathogen may be a
Trichostron~ylus col~briru""is or Trichosl,ol,~/lus ~i. The Fasciol~ species
15 may be Fasciol~ he~,dlica or F~r~ iQ~
In a preferred ~spect the sample of the dise~se pathogen may be taken
at a stage of develû~.",e,ll during which it is thought to be most susceptible to
attack.
It is postulated that the time at which the ~ise~se pdll,ogen sample is
20 taken is important since a ~ arasile is vulnerable for only a short time after
entering the subject after which it may change structure and is no longer
vulnerable to immune attack and may no longer express protective anliyens.
For example in the cases of the dise~se ~,~II,Gyens F. he~ali~. Q.
circumcincta and I. colublironl,is it may be suitahle to take the sample from the
2s larval stage.
The animal from which the bicl~içal san,ple may be taken may be of any
suitable type. The animal from which the bio'cgic~l sample is taken may be an
immune animal. The biological sample may be taken a short time after the
immune animal has been challenged with a pathogenic infection. The animal
30 may be an animal such as sheep or cattle.
The bioloyical animal sample may be of any suitable type. The biological
sample may be from animal tiss~es organs blood Iymph or Iymph nodes. The
biological sample may be taken from any section of the i"rectecl animal.
_
WO 9S/09182 217 2 ~ ~ 2 6 - PCTIAU94/OOS73
However, it is preferred that the sdlllpl?s be taken from the i"re~;tecl site or an
area of a lesion which may be fo""ecl in certain cliso-~scs or an area close to or
draining from the i"rected site or a lesion such as in the Iymph nodes. P,eferdbly
samples may be taken from the h~palic Iymph node, abGn,asal Iymph node or
s ",ese"l~ric Iymph node. Serum/plas",a sarl,~lcs are not preferred as the
biological samples. It has been found that the majority of antibodies found in aserum/plasma sample are irrelevant to pr~lection or specific diaynosis of a
pathogen or are u,)~lated to the p~ll,Gyen.
In col,l,ast, the probes used in the present invention are highly ellricl1ed
lo in pathogen-spe~iric antibodies and can be selec~d to be restricted to the
pathogen-stage of particular i",po, lance to protective immunity.
The cells isol~terl from the biological sample may include B cells. The
cells may be isolated similarly at a time known to include a secr~tion and/or
antibody producing period. All~r"dlively, the cells may include memory cells
which may be generated at a later stage in certain dise~ses.
Thus, pl~ferdbly the cells are taken a short time after vivo stimulation,
preferably within appro~i",dt~,ly 2 to 13 days II,erecner with, for example, therelevant parasite stage II,er~by resulting in the in vivo induction of antibody
forming cells which will secl~te specific antibodies into the culture medium after in
vitro incubation. No, or very few antibodies may be secreted in culture medium
without prior in vivo stimulation of resting Iymphocytes.
In vitro secrelio" of ar,libodies in the culture medium by recently activated
B cells may be enhanced by the addition of helper factors to the cultures. The
helper factors may be cytokines used alone or in combination, including
interleukin 1, 2, 3, 4, 5, 6, 7 and 8, colony stimulating factors, interferons and any
other factors that may be shown to have an enhancing effect on specific antibodysecretion by B cells.
The method of producing the antibody probe may include a further step of
activating the cells isolated to prulirel~le and secrete and/or release antibodies.
The cell activation step may include adding a cell activating agent to the
culture medium. The cell activating agent may be parasite-derived or may be
selected from mitogens and helper factors produced by leucocytes, or their
synthetic equivalents or co",bi"aliG,)s thereof.
WO 95/09182 2 3i ~ 2 4 Y ~ PCT/AU94/00573
The mitogens may be sele;le~l from products derived from pol~ewccd
(Phyto'~cca ~rnericarl~.) also known as pokeweed n.itùgel, (PWM),
phorbolmyristic acid (PMA), polyvinyl-pyrrolidone (PVP), polyadenylic-polyuridylic
acid (poly(A-U)), purified protein derivate (PPD), polyinosinic-polycytidilic acid
5 (poly(l-C)), lipopolysaccha. ide (LPS), staphylococc~l organisms or products
thereof, Bacto- sl-eptolysin O t:age,)l (SLO), Staphylococc~l phage Iysate (SPL),
Epstein-Barr virus (EB\/), Nocar~lia water-soluble ,..itogel. (NWEM), phytohema-agglutin (PHA) Concanavalin A (Con A) and dextran-sulphate and mixtures
thereof. The cell prolireralion agent may be any agent that indirectly or directly
lo results in B cell proli~erdliol1 and/or antibody secretion such as solid-phase anti-
immunoglobulin. The helper factors may be cytokines including interleukin 1, 2,
3, 4, 5, 6, 7 and 8, colony stimulating factors, i,.l~ireru.,s and any other helper
factors that may be shown when added alone, or in comiJi, dlior with other factors
and agents to have an enhancing effect on speciiic B cell prc!;rerdliol) and/or
lS antibody secretion. This in no way is meant to be an exhaustive list of mitogens
and cell activating agents including helper factors.
The in vitro culturing of the cells may be conducted with or without prior
steps to separate sub-populalio"s of cells. The harvesting of al-lilJo.lies may be
conducted by harvesting of the super,-ala,-l from the culture medium. This
20 supernatant contains a,.lil,odies secreted by these cells during the in ~i~ culture
or artificialiy released from the B cells, for example by Iysis of the B cells. It has
been found, su-,uri~i"yly, that the antibody-containing su,uerlldldnl~ may be used
directly to detect antigens of a pdll,oyen.
In a preferred aspect, the sample of liise~se pdll-ogel- may be mixed with
25 a standard buffer solution and placed on a slandar~l support such as an SDS-
polyacrylamide gel to separate the pr~,teios co,-tdi.-ed ll-er~i,-. The separdled
proteins may then be lldn:,rerl~d to nitro-cellulose, nylon or other sheets.
The corresponding antibody probe produced as desc,iLed above may be
utilised simply in the form of the su~Jellldldnl harvested from the culture medium.
30 Alternatively, the antibodies may be separdted and purified.
The anliL,ody cGIlldilled in the culture medium may be used for
purification of the antigen. An affinity p~-,ircdliG~ ,referdiJly immuno-affinity
puliricdlion may be used.
~ ~ ~2~2 ~
WO 95/09182 PCT/AU94/00573
-- 8--
The antigen loc~led as descriL.ed above may be dele~e~l utilising any
suitable assay techniq~e.
The antibody probi"g step may accordi"gly further include subjecting the
product produced thereby to a clete~1iG" assay.
The dstection assay may include Western blot techniques. The detection
assay may be an immunoprecipitation assay a radioimmu"oassay an enzyme-
linked immunoassay or immunofluor~sce"l assay.
Accordi"gly the auli~ens may be purified by a Illelllod which includes
providing
a crude antigen mixture;
an antibody against a ciise~se pathogen selected from Fasciol~.
Osterta~ia and Trichosl,uns~ylus~ and related species, immobilised on a
suitable support;
subjecting the crude a"ligen mixture to an afffinity ch~ol"alography
15 utilising the i"""oLilised antibody; and
isolating the purified antigen so ~""ed.
The aulibody can be obtained from the culture supernatant probe
described above by conventional Ill~lhods. For example methods usually used to
purify irnmunoglobulins from serum or plasma e.g. precipitation with ammonium
20 sulphate fra~;tionalion with caprylic acid ion exchange chromatography or by
binding and elution from immobilised protein G or protein A may be utilised.
Antibody so obtained can then be coupled to suitable supports e.g.
CNBr-activated Sepharose 4B (Pharmacia) Aff-gel (Bio-Rad) or other afffinity
chlo,oatography supports able to bind proteins.
Immobilised antibody can then be applied to the fractionation and
purification of specific antigen from a complex parasite extract by affinity
chromatography. After binding of antigen to imrnobilised antibody unbound
macromolecular species can be washed away from the solid support with e.g.
buffers containing 1.5M NaCI. Subsequently the antigen can be eluted from the
30 affinity column with e.g. Iow or high pH buffer or buffers containing chaotropic
ions e.g. 0.~ - 3.0 M sodium thiocyanate.
The anligel)s isolated or located may be used in the preparation of
monoclonal antibodies.
W O 95/09182 2 1 7 ~ ~ ~ 2 PCT/AU94/00573
Accordingly the ~,resent invention further provides a method for
producing a monoclonal antibody aydi"~t an antigen of a disease pathogen as
described above which method inol-~des
~ . .
provldlng
a B cell capable of producing ar,lil-od;es against said protective
antigen or r,ay,nenls II,er~of and obtained from an animal immunised
with a protective allligell againsl the disease p~lhoyen as descriLed
above; and
a myelG",a cell;
fusing the B cell with the myelo",a cell;
propagating a hyLrido",a ro,lllecl thereby and
harvesting the ar,liL,o.ly produced by said hybridoma.
The ",ol,oclol)al antibodies may form the basis of a passive l,~:dl",ent of
the disease discllssed above.
1~ Plt:reldL,lythe antigen is a F~sciol~ hev~lica a~li3ell.
The monoclonal arili60dies so rc,r",ed may be sel~ le~ from the group
consisting of FY 4-7-12, FY 3-3-1 F~Y 3-3-2, FY 3-5, FY 4-7-6 and FY 1-6 as
hereinafter described.
Having idel,li~ied the auligen(s) molecular biology or chemiGal techniques
20 e.g. cloning techniques may be used to produce unlimited amounts of this antigen
or alternatively synthetic peptides cGr,t:s~ onding to different fragments of the
identified antigens may be used as a means to produce a vaccine.
Accordingiy in a pr~:fe, l~d aspect of the present invention there is
provided a r"ell,od for pr~pdri"g a sy"U,etic antigenic polypeptide agai"sl a
25 disease pathogen selected from Fasciol~. Oslt:,la~ia and Trichosl,u"~ylus
species and related species which ",ell,od incl~ es
providing
a cDNA library or genomic library derived from a sample of a
dise~se pathogen selected from F~crirJla. Osterta~ia and
Trichostron~ylussperies and related species; and
a col,espol,di"g antibody probe including
at least one antibody against the respective rlise~se pathogen
produced by a method including providing a biological sample
WO 95109182 ~ ~ ~ 2 4 ~ ~ PCT/AU94100573
- 10-
from an immune animal taken a short time after the immune
animal has been challenged with a pdll,ogel) or pathogen
extract s~le~,1erl from F~ ciola. Osle,Ld~ia and
Tlichosl~ ylus species, and related Spe~'ES, or
S a cor,espo"diny monoclonal antibody derived therer,u"" or
a polyclonal or ~GnoclGI)al antibody generated after injection
of the purified anliyen;
generating sy"ll,elic polypeplides from the cDNA library or genomic
library;
probing the s~",ll,~lic poly~.eplides with the antibody probe; and
isolating the sy"ll,t:lic ai,li53enic poly~,eplide detec~i thereby.
Either cDNA or genomic libraries may be used. The cDNA or genomic
libraries may be asserl,bled into suitable exp,~ssiG" vectors that will enable
transc,i~.lion and the suhse~uent expression of the clone of DNA, either in
prokaryotic hosts (e.g. bacteria) or eukaryotic hosts (e.g. ma"""alian cells). The
probes for screening the libraries may p,~ferably be S~IECted from:
(i) synthetic oligonuclaotide probes based on the amino acid sequence of
the antigen idel ,liried and purified as described above;
(ii) PCR products generated from the synthetic oligonucleotide probes;
20 (iii) antibodies based on synthetic peplides derived from amino acid
sequence data of the antigen identified;
(iv) antibodies obtained from the culture medium produced as described
above;
(v) monoclonal or polyclonal antibodies produced against the antigens
identified and purified as described above; and
(v) recombinant or synthetic ",G"oclonal antihodies or polypeptides with
specificity for the antigen, e.g. as descriL.ed by Ward et al 1989, Nature
;~, pages 544 to 546.
Preferably the cDNA library is derived from a sample of Ostert~gia
30 circ-""ci~ : the cGr,esponding antibody probe is a r"GnoclGnal antibody raised
against the 32-36 kD doublet antigen.
In a still further aspect there is provided a synthetic antigenic polypeptide
prepared as described above.
-
~7~2
WO 95/09182 PCTIAU94/00573
- 11 -
The synthetic a"liyel)ic polypeptide may be selected from clones 3-2 and
5-2b, having the amino acid sequence
M.A.FETNYP IPYRSKLTEP FEPGQTLTVK GKTGEDSVRF TINLHNSSAD
FSGNDVPLHV SVRFDEGKIV CNSFAKGEWGKEERKSNPYK KGDDIDIRIR
s AHDSKFQIFV DQKELKEYEH RLPLSSITHF SIDGDVLITH IHWGGKWPV
PYESGLAGEG LSPGKSLYLY GMPEKKGKRF HINILKKNGD IALHFNPRFD
EKAWRNSLI SNEWGNEERE GKMPFEKAVG FDLEIKNEDY PFQIMVNGER
FASYSHRLEP HELNGLQIGG DVEITGIQLH
, as l,erei,~dller desc,ibed.
Accordingly in a further aspect of the present invention, there is provided
a putative protective antigen agai"sl a dise~se pathogen selected from FPSCjQIa.Ostertagia and Tricho~l,un5Jylus species, and r~ldled species, prepared by a
method including
providing
a sample of a liseAse pathogen sel~,le.J from Fasciola. Ostertagia
and Trichostrongylus species, and related species; and
an antibody probe including at least one antibody ayai"st a disease
pathogen selected from Osterta~i~ and Trichostrongylus species, and
related species, produced by a method as described above;
probing the dise~se pathogen sample with the cGr,esponding antibody
probe; and
isolating the protective a"liyen detected.
The protective antigens may function as vaccine and/or diagnostic
antigens as discl~ssed below.
In another aspect of the present invention, there is provided a monoclonal
or polyclonal antibody against a plotecti~e antigen, or ~,dy,nent thereof, against a
disease pathogen selected from F~cciol~ Osle,I~Qja and Trichostrongylùs
species, and related species.
In another aspect, there is provided a monoclonal or polyclonal antibody
against a protective a"Ligen as herei.lbef~re described, or fragment thereof,
against Fasciola species, and related species.
In another ~-spect the present invention provides a ",etl,od for preventing
infection cause~ by a disease ~ dll,o~en selected from F~ , Ostertagia and
WO 95/09182 ~ ~ 7 ~ 4 ~ 2 12 - PCT/AU94/OOS73
Tlichosl,u,l~ylus spe~-es, and related species, in animals, which nl~lhod incl~ es
ad~ islering to an animal an effective amount of at least one protective antigenas described above.
rlererdbly, the ~.r~tel~tive antigen is an a"li~e" derived from F~c~ la
s hep~tica or Tricho~lrony~lus colubriru~nis~ as herein described.
In a still further aspect of the present invention there is provided a method
for the l,~dl",enl of inre~;tion c~used by a disease pathogen selected from
F~ciola. Ostert~ia and Tlicho~t,ul,.Jylus species, and related species, in
animals, which Illetllod includes adn,i"isteli"g to an animal a therapeutically
effective amount of a monoclonal or polycional allliLo.ly to a protective antigen as
described above.
The present invention further provides a vaccine or veterinary
composition including a prophyl~ctic~lly effective amount of at least one antigen
against a dise~se pathogen selecled from F~-cciola. Osterta~ia and
Trichostron~ylus species, and related species, as described above. Preferably
the vaccine co,nposilion includes a plurality of protective antigens against a
number of disease pathogens.
The present invention further provides a vaccine or veterinary
composition including a therapeutically effective amount of at least one
monoclonal or polyclonal antibody against a protective antigen as described
above. Preferably the vaccine composition includes a plurality of monoclonal or
polyclonal antibodies.
In the preferred forms, multiple protection may be provided to an animal
via a single treatment.
The vaccine or veterinary compositions according to the present invention
may be administered orally or may be administered parenterally (for example by
intram~scul~r, subcutaneous, intradermal or intravenous injection).
The amount required will vary with the antigenicity of the active ingredient
and need only be an amount sufficient to induce an immune response typical of
existing vaccines.
Reactive experi,,,el)taliûl) will easily establish the required amount.
Typical initial doses of vaccine or veterinary compositions may be approximately0.001-1 mg active ingredienVkg body weight. The dose rate may increase or
WO95/09182 ~ ~ 7 ~ 4 ~ ~ PCT/AU94/00573
- 13-
multiple doses may be used as needed to provide the desi,~d level of ~.rote.:lion.
The vaccine or veteri"ary cG"~posilion accor~li"g to the present invention
may further include a v~terin~,y acce,i~lable carrier, diluent or excipient thereof.
P,eferably the active ingredient may be suspei)ded or dissolved in a carrier. The
carrier may be any solid or solvent that is non-toxic to the animal and co"~,aliL,le
with the active ingredient. Suitable car,ier:~ include liquid carriers, such as normal
saline and other non-toxic salts at or near physiological conce"l,dliol,s ànd solid
carriers such as talc or sucrose. Adjuvants such as Freund's adjuvant complete
or incomplete, or immu"Gr"odulators such as cytokines may be added to enhance
the antigenicity of the antigen if desi,~d. When used for admi"islari"g via the
bronchial tubes, the vaccine is suitably presented in the form of an aerosol.
The vaccine or veterinary col"position accordiny to the present invention
may be incorporated into a live vector (eg. vaccinia virus salmonella) or
administered as DNA or RNA as described in Tang et al., Nature 356: 152 1992.
s In a still further aspect of the present invention there is provided a
diagnostic assay kit including a diagnostic anligel) or fragment thereof against a
disease pathogen identified and purified as described above.
The diag"oslic kit may be lltilised to detect i"r~ctior,s in animals caused
by a disease pathogen selectecl from O. circ--" ,ci, Icld F. hep~tica
colubriforrnis or related parasites.
The diagnostic assay kit may be utilised in conjunction with a diagnostic
assay. The diagnostic assay may include Westem blot techniques. The
diagnostic assay may be a diagnostic immunoassay. The immu"oassay may be
an immunoprecipitation assay a r~di i. "murioassay an enzyme linked
immunoassay an immunofluor~scel)l assay or a chei"ih~minescent assay.
The present invention will now be more fully described with reference to
the following examples. It should be under~ood, however that the descri~ lio
following is illustrative only and should not be taken in any way as a ~ L,i.;lion on
the generality of the invention described above.
IN THF FIGUPFS:
FIGU~F 1a: Osterta~ia circu",c;,l.;ld
SDS-PAGE (12.~% gel) and Western blot analysis of L3 larval
extracts of Osl~:, Ld~ia circumcincta ,~)lul~ed with Iymph (1/1000
WO 95/09182 ~ PCI/AU94100573
- 14-
dilution) from experimentally immune Suffolk lambs. Two clusters of
immunoreactive species of apparent M.W. 26-36 and 95-105 kD
were detected. The same regions were also identified with culture
supernatant of abomasal Iymph nodes from challenged sheep.
S FIGU~F~ 1 b-1 e:
Reaction of clones with ~ti-doublet mf~h or sheep serum by ~laque
immunoassay
IPTG filters lifted from plates of each clone were cut into pieces and
reacted with antibody. Detection was with alkaline phosphatase
lo conjugated anti mouse IgM+lgG or anti-sheep IgG. Filters were
reacted with Fig. 1b: anti-doublet mAb, Fig. 1c: a negative control
IgM mAb, Fig. 1d: sheep serum raised to purified doublet antigen,
Fig. 1e: negative control sheep serum. Clones shown are 7-1, 7-2,
5-2b (2 isolates), 3-2, 8-2 (2 different dilutions) or negative control
plaques.
FIGUI~F 1f: Western ~lot of O.circumcincta 1.~ extract probed with ar,~i~o.lias affinity
purified from clones
Samples of aqueous extracts from L3 larvae were electrophoresed
on a 12.5% SDS-polyacrylamide gel, which was then electroblotted.
The Western blot was reacted with affinity purified antibodies eluted
from plaque immunoassays, with mAbs, with sheep sera or with
abomasal Iymph from a repeatedly infected, immune sheep, and
reactions detected with alkaline phosphatase anti mouse IgM+lgG
or anti-sheep IgG. Lane 1: negative control mAb; 2: anti-doublet
mAb; affinity purified antibodies eluted from 3: clone 7-1; 4: clone 7-
2; 5 and 6: clone 5-2b (2 isolates); 7: clone 3-2; 8: clone 8-2; 9:
negative control plaques; 10: sheep anti-doublet serum; 11:
negative control serum; 12: Iymph from immune sheep; M: pre-
labelled molecular weight markers (Biorad). The position of the
doublet is arrowed.
FIGU~!F 1g: Nucleotide and predicted amino acid sequence of clones 3-? and 5-
~h
Amino acids are shown in the 3 letter code.
}t~' l~l~ ~i~l (Rulc 91)
wo 95/09182 ~ 1 7 2 4 ~ 2 PCT/AU94/00573
FIGU~F 1h: Ali~"r"enl of O.circumcincta clones 3-7 ~nd 5-~h predicted amino acid sequence with G~Ps from O.volvulus ~nd C.~ ans.
Amino acid sequences for the GBPs were extracted from datah~ses
using ANGIS (O.volvulus from GenPep ~t~h~se, accession no.
U04046_1; C.eleg~rls from PIR ~t~h~se, ~ccession no. S27798).
Single letter codes for amino acids are shown.
FIGU~F 1 i: Demon~l,dlion that the doublet antigen is a lectin-like GBP.
O.circumcincta L3 larvae were extracted with buffer and samples
were applied to an asialofetuin-Affigel 15 column. After washing,
bound protein was eluted with 100mM lactose. Lanes: M: molecular
weight markers; 1: L3 extract; 2: last flow through fraction from
column; 3-7: lactose elution fractions. The position of the doublet
antigen is arrowed. (12.5% SDS-PAGE, coomassie stained).
FIGUI~FS 1j and 1k:
Fs~ ssion of recG"~b.. ~ar,l a,ltigens in F coli.
Figs. 1j and 1 k. CTAB-solubilised inclusion bodies were
electrophoresed on a 13% gel which was then electroblotted. Half of
the Western blot was reacted with anti-doublet mAb and detected
with alkaline phosphatase conjugated anti-mouse IgG+lgM (Fig. 1j)
and the other half was reacted with sheep antiserum to purified
doublet and detected with alkaline phosphatase conjugated anti-
sheep IgG (Fig. 1k). Lane 1: clone 7-1; 2: clone 7-2; 3: clone 3-2; 4:
clone 8-2; 5: clone 5-2b; 6: pMOSELOX control.
FIGU~F~ 7~ . - O~ Idg;a circumcincta
Mean eggs per gram faeces (epg) of vaccinated (.--.) and control
(o--o) sheep after infection with L3 larvae in three separate trials.
Fl~;UI2F ~: Mean faecal egg counts of control and vaccinated sheep used in the
third Osterta~ia vaccination trial after heterologous i,~r~;tiG" with H.
contortus.
30 FIGU~F 3: Tricho~l,onyylus colu~rir~,r",is
Western blot of 3 species of L3 nematode larval antigens probed
with MLN supernatant of T.colubriformis infected sheep.
Lane 1. Bio-Rad prestained mol~clll~rweight markers.
WO 95/09182 ~17 ~ 4 ~ 2 PCTIAU94/OOS73
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Lane 2. O. circumcincta L3 antigen.
Lane 3. H. contortus L3 antigen.
Lane 4. T. colubriformis L3 antigen.
Brackets = antigen of Trichostrongylus colubriformis
5 FIGUPF 4: Fasciola hepatica
Western blot of NEJ fluke antigen probed with supernatant from
hepatic Iymph node (1), mesenteric Iymph llode (2), and spleen (3)
7 days after oral challenge with 200 Mc of previously infected and
cured rats.
Lane 4: Bio-Rad Prestained molecular weight markers.
Arrow = position of ~200 kD antigen recognised only by the MLN
supernatant.
FIGURF 5: Fasciola hepatica
Western blot of NEJ antigen probed with supernatant from hepatic
Iymph node (5), mesenteric Iymph node (6) and spleen (7) of twice
immunised and cured rats challenged with 400 Mc.
Lane 4: Bio-Rad prestained molecular weight markers.
Arrows - position of antigens recognised only by the MLN supernatant.
FIGUF~FS 6a-6d: - Fasciola he~atica
Indirect immunoperoxidase staining of NEJ flukes with (Fig. 6a)
mcAb 38.27 against sheep MHC class ll (negative control) X 40.
Similar negative staining was observed with mcAb's FY3-5 and FY1-
6, (Fig. 6b) mcAb FY3-3-2,X40; (Fig. 6c) mcAb FY3-3-2, X 100 and
(Fig. 6d) mcAb FY3-3-1, X 100. Note the strong reticular type
2s staining in (Fig. 6b) and (Fig. 6c) compared to the more restrictedspeckled staining in (Fig. 6d). The arrows point to the oral sucker of
the NEJ flukes.
FXAMPI F 1
OSTFRTAGIA CIRCUMCINCTA
30 Parasites and Fxperimental Animals
O. circumcincta third stage larvae (L3) were collected from faecal cultures
of donor sheep experimentally infected with the parasite. Immune animals were
obtained by repeatedly infecting sheep with O. circumcincta larvae and then
-
~7~4!~2
WO 95/09182 PCT/AU94/00573
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monitoring faecal egg output. When a challenge dose produced few or no eggs
in the ~aeces the animal was said to be immune. Once immune the sheep were
drenched with IVERMECTIN and left for a period of at least four weeks before
being challenged with 60 000 L3 larvae and then killed five to eight days post
s challenge.
Preparation of Culture Supe",~la,lts
AbGn,asal Iymph nodes (ALN) were removed and cell suspension
prepared as desc,ibed in Australian Patent No. 640364 r~f~:r,~d to above. Bulk
cultures of 10-50 ml were set up in culture flasks (Miles) at a conce"l,~lio" of 0.5-
1.0 x 107 cells/ml in DME + 10% foetal calf serum. Preliminary ex~eri",enl.
established that most of the antibodies in the culture s~" er"dlant were produced
by the al,Li~ody secreting cells present in the in vivo stimulated Iymph nodes and
that this was not further i"cr~ased by stimulation with pokeweed mitogen (PWM).
PWM was ther~:tore not added to further cultures and culture supernatants were
ls harvested after a five day inc~h~tion of cells at 37C in 5% CO2 atmosphere
then stored at-20C until used.
Cannul~tion of ~hom~c~l Iymph nodes and collection of Iym~h
Sheep (Suffolk lambs) were rendered immune as described above
challenged with 60 000 L3 larvae and the con""o,) abomasal Iymph duct
20 cannulated 4 days after challenge. Lymph was collected for several days aftercannulation and the cell-free Iymph stored at -20C prior to use.
Preparation of Antigens for SDS-PAGF ~nd Western blots
Third stage larvae of O circu,n, ;,)cla were exsheathed in approximately
0.5% NaHOCI in a CO2 enriched ~t",osphere for 20 minutes at 37C to remove
25 the second stage sheath. The larvae were then repeatedly washed and
centrifuged at 3 000 g for 10 minutes in phosphate buffered saline (PBS) pH 7.4. After the sixth wash they were tra" .ter,~d to 500 ml of DME medium pH 6.8 in the
presence of 200 U/ml penicillin and 0.2 ug/ml sl,e,~)ton"~cin and cultured at 39C
with 20% CO2 in air for 3 days. The culture media was then centrifuged at 3 000
30 9 for 15 minutes at 20C and the pelleted in-vitro switched L4 larvae stored at -
70C.
Antigens were extracted from exsheathed L3 in-vitro switched L4 ir~-vivo
WO95/09182 2; ~ 2 l8- Pcr/Au94/00573
L4 and adult stages by freeze thawing 3 times, then homogenisation using a
polytron homogeniser (Ki!~m~i~ca GmbH, Swikerland), exl,a-;lion over-,iylll in
50mM Tris HCI pH 8.0, 150mM NaCI, and centrifugation at 50,000 xg for 30
minutes. The super"dta,.l co..lai..i..g solubilised antigens was stored at -70C.
The e~cl-dct~cl antigens were run under non-reducing conditions on 12.5%
(WN) SDS-polyacryld",i"de gels and western blotted onto PVDF membrane
(Immobilon, Millipore) or nitrocellulose.
Preparation of ~nti~ens for v~cci, l,.li~l l tri~lc
Third stage larvae of O. circu",ci"ctd were exsheathed in CO2 enriched
lo atmosphere for 2-3 hours at 37C to remove the second stage sheath. The
exsheathed L3'S were freeze-thawed three times, then homogenised using a
ground glass homogeniser followed by overnight extraction in 150mM NaCI,
50mM Tris pH 8.0 conlai"ing 2% (w/v) Hexadecyll,i"-ell,yl- ammonium bromide
(CTAB, Sigma). Particulate matter was removed by 30 min centrifugation at
3009. The supe"lata,)t was further centrifuged at 15,000 xg for 30 min and the
soluble extract stored at -20C.
The extracted antigens were run under non-reducing conditions (without
boiling) on 10% CTAB-acrylamide gels. The appropriate area of gel was
identified by western blotting 2 strips on either end of the gel and reacting the
20 strips with positive Iymph. The gel area corresponding to the immuno-reactiveregion was excised, mashed and incubated in 2% CTAB solution overnight to
passively elute the anligens. The CTAB was removed by incubation with Dowex
resin in 2M urea at pH 10 for 15 min, then dialysed against PBS overnight to
remove the urea. The antigen was concentrated in Centriprep concel,t,alor
25 (Amicon), the protein conce"l,dlion determined using BCA assay (Pierce) and
used to immunise sheep. This antigen preparation was shown to contain the 26-
36kD immunoreactive region when run on SDS-PAGE gels.
Idenliricalion of Antigen
Western blotted antigen preparations were probed with in vitro culture
30 supernatant from ALN and Iymph from cannulated abomasal Iymph nodes from
infected sheep. Both the culture supernatant and the Iymph highlighted two
regions of molecular weights 26-36 and 95-105 kD (see Figure 1).
WO 95/09182 ~ 3~724 ~ 2 PCT/AU94/00573
- 19-
lncubation of the blotted antigens with lectin conjugates revealed that the
antigens were able to bind some lectins. This indicates that some of the antigens
are glycosylated.
Protein Sequencing of the ~6-36kn Antigens Identified by Western Rlottin~
s The N-ter",i"al amino acid sequences of the antigens within this region
were determined after separating the proteins by SDS-PAGE, followed by
electrophoretic transfer to ProBlott sequencing membrane using CAPS buffer.
The transferred proteins were loc~ted on the membrane by staining with
coomassie blue and were excised. The sequence was determined in an Applied
Biosystems 476A protein sequencer fitted with a blot cartridge. Further internalsequence data were generated by digesting the CTAB purified antigens with
cyanogen bromide, separating the fragments by SDS PAGE, blotting onto ProBlot
and sequencing as above. The results of the protein sequencing are summarised
below. Two bands in the upper part of the 26-36kD region did not give any
sequence and were presumably blocked at the N-terminus. These two bands in
the upper 32-36kD region are further referred to as "the doublet" since a
monoclonal antibody generated against this region recognises both bands (see
Fig. 1~ indicating that they are similar molecules.
The sequences obtained from the lower bands in the 26-36 kD region
were screened against the sequence database libraries available at the
Australian National Genomic Inror",dlion Service, using the program FASTA. The
results are shown below. Two of the molecules could be identified by virtue of
their high degree of hor"oloyy to sequences in the d~t~hanks. The homologous
sequences were tropomyosin and glutathione S-transferase.
The O. circumcincta sequences homologous to
(A) tropomyosin are:
1. N-terminal sequence: MKAEEVRQALK
2. Internal sequence: VEADLERAEERAEAAGENKWVL
(B) Glutathionine S-transferase:
N-terminal: VQYKLWFDGRXAAEV
To obtain sequence from the region containing the doublet bands, an
aqueous extract of homogenised third stage Ostertagia circumcincta larvae was
prepared and the proteins in the extract were separated by SDS PAGE under
RECTIFIED SHEET (RULE 9~)
WO 95/09182 ~ ~ ~ 2 ~ 9 2 , PCT/AU94/00573
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reducing cond;liGIls. The doublet bands were excisecl from the coomassie blue
stained gel and the proteins were then extracted from the excised gel pieces by
electroelution. The isol~ted protein was digested with Trypsin and the peptides
thus generated separdled by high pe, rul " ,ance reversed phase liquid
5 chro" ,alography (HPLC). The purified peptides were sequenced by Edman
degradation in an Applied Biosystems 476A protein sequencer. The following
peptide sequences were determined
1 ) SAHGPPGQ
2) FPHGPSYQHGYA
3) IVTHPNR
Cloning of cDNAs encodin~ the doublet ~ntigen
I ihrary Preparation
RNA was extracted from freshly harvested stage L3 larvae, which had
been snap-frozen in liquid nitrogen (Chomczynski & Sacchi, 1987, Anal.
Biochem., 162, 156-159). Messenger RNA (mRNA) was isolated by oligo dT
affinity ch~clmdloyldphy on mAP paper (Amersham Australia). Double-stranded
complementary DNA (cDNA) was prepared by pri",ing 2~9 mRNA with either
oligo dT or random primers using a cDNA Synthesis System Plus kit (Amersham
Australia). Oligo dT and random-primed cDNA was pooled and EcQRI adaptors
20 were added (cDNA rapid adaptor ligation module, Amersham Australia), and the
adapted cDNA ligated to E~QRI cut, de-phosphorylated bacteriophage expression
vector ~MOSELOX arms (Amersham Australia) and packaged using the ~-DNA
vitro packaging module (Amersham Australia). A primary library of 1.4 x 106
plaque forming units (pfu)/mL, of which > 90% were recombinants, was obtained.
2s The library was amplified on E.5~Qli ER1647 cells before use.
I ihrary Screening
5 x 105 pfu of the amplified cDNA library were plated out on E.sQli
BL21 (DE3)pLysE cells at 5 x 104 pfu/plate. When pin-prick size plaques appeared(after 4-6 hours at 37C), the plates were overlaid with nitrocellulose filters which
30 had been impregnated with 1 OmM isopropylthio-~-g~lactosicle (IPTG) and
incubated for a further 6 hours at 37C. Plates were then stored overnight at 4C.
Filters were removed from the plates and washed in TNT (10mM tris-HCI, pH 8,
150mM NaCI, 0.05% Tween 20), blocked in BLOTTO (5% w/v low fat milk powder
WO 95/09182 - 21 - PCT/AU94/00573
in TNT) and incubated for 2 hours at room temperature with an IgM mouse
monoclonal antibody (mAb) (undiluted culture supernatant) which had been
raised to the doublet antigen. After washing in TNT, filters were inc~h~tecl with
alkaline phosphatase conjugated rabbit anti-mouse IgG+lgM (Jackson
S Immunoresearch) at a 1:5,000 dilution in BLOTTO for 1 hour at room temperature.
After further washing in TNT, filters were developed with 0.165mg/mL 5-bromo-3-
chloro~-indolyl phosphate (BCIP) and 0.33mg/mL nitroblue tel,d~olium (NBT) in
alkaline phosphatase buffer (0.1M tris-HCI, pH 9.5, 0.1M NaCI, 5mM MgCI2).
Fifteen putative positive plaques (some of which were very faint) were picked and
10 re-screened with the monoclonal antibody as described above, after which 5
plaques remained positive. These plaques were tertiary screened and amplified
stocks prepared by plating on ER1647 cells.
Analysis of clones
To determine the specificity of clones for the doublet antigen, plaque
15 immunoassays were performed. Plaque-purified clones were plated out on
BL21(DE3)pLysE E. coli cells and induced with IPTG filters as described above.
The filters were then reacted with the anti-doublet mAb, an unrelated IgM mouse
mAb, and with antiserum raised in sheep to purified doublet antigen or with
negative control sheep serum (both sheep sera were used at a 1:50 dilution in
20 BLOTTO, and the secondary antibody was alkaline phosphatase conjugated rabbitanti-sheep IgG from Jackson Immunoresearch at 1:5,000). Positive reactions
were detected as described above. All clones were positive with the anti-doubletmAb and negative with the unrelated mAb and with negative sheep serum. Two
clones, designated 3-2 and 5-2b, were strongly positive with the anti-doublet
25 serum raised in sheep (Figures 1b-1e).
Clones, or ~MOSELOX control plaques, were plated out at 2,000 plaques
per 80mM plate to achieve confluent Iysis. As soon as pin prick plaques
appeared, IPTG filters were added and the incubation continued overnight. Filters
were washed extensively with TNT, blocked 1 hour in BLOTTO and incubated 4
30 hours at room temperature with the anti-doublet sheep antiserum (1 :50 in
BLOTTO). (Before use, the serum was depleted of anti-E~Qli antibodies by
incubation with filters from plates of wild-type ~MOSELOX plaques.) Filters werethen washed 5 times in TNT, once in borate wash buffer (0.1M boric acid, 0.5M
~ ule 91)
~172~g~
WO 95/09182 PCT/AU94/00573
- 22 -
NaCI, 0.05% Tween 20, pH 8) and once in PBS (140mM NaCI, 2.7mM KCI, 8mM
Na2HPO4, 0.0015mM KH2 P04). Bound, affinity-purified antibodies specific for
each clone were eluted for 1 minute in 0.1M glycine, 0.15M NaCI, pH 2.6, 5 mL
for each filter and i",medialely neutralised by adding to tubes containing 300,u1 of
s 1 M tris-HCI, pH 8. The antibodies were dialysed against TNT for 1-2 hours at 4C,
low-fat ~milk powder added to 5% w/v and stored at -20C. Aqueous extracts of L3larvae were electrophoresed on a 12.5% denaturing SDS-polyacrylamide gel and
proteins transferred to Irnmobilon membrane (Millipore) by electroblotting. The
membrane was washed in TNT, blocked in BLOTTO and cut into strips. Strips
were incubated with the affinity purified antibodies from the clones, with anti-doublet mAb or negative control mAb, or with sheep anti-doublet antiserum or
negative control sheep serum at 4C overnight. Detection was with alkaline
phosphatase conjugated anti-species antibody followed by colour development
with BCIP and NBT. Antibodies affinity purified on the 2 clones which were
positive with sheep anti-doublet antiserum, 3-2 and 5-2b, specifically recognised
the doublet band from the O.circumcincta larvae on the Western blot (Figure 1fl.The clones were rescued into the plasmid form (in pMOSELOX) by
plating on E~Qli BM25.8 cells in the presence of carbenicillin as recomn)ended by
the manufacturer of the vector (Amersham Australia). Plasmid DNA preparations
were performed by alkaline Iysis and CsCI density gradient as described in
'Molecular Cloning, A Laboratory Manual, second edition' (Sambrook, J., Fritsch,E.F. & Maniatis, T., 1989, Cold Spring Harbor Laboratory Press). Samples of
plasmid DNA were digested with E~QRI and analysed on a 1% agarose gel in
TAE buffer (40mM tris-acetate, pH 8, 1mM EDTA) containing 50~g/mL ethidium
bromide. Clone 3-2 contained 3 ~QRI fragments of approximately 1000, 400 and
200 base pairs, while clone 5-2b contained 2 fragments of 1000 and 400 base
pairs.
nNA Sequencing
DNA sequencing was performed by the dideoxy method using a
Sequenase kit accor~ lg to the manufacturer's instructions (United States
Biochemical Corporation). Sequencing reactions were performed in the presence
of a-35S-dATP and gels autoradiographed. Initially, primers based on the vector
~ 1 (Rule 91)
WO 95/09182 ;~ ~ 7 ~ 4 ~ 2 PCT/AU94/00573
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flanking the insert were used (T7 gene 10 and SP6 primers). Further primers to
sequence the length of the inserts were designed based on the sequence
obtained. Clones 3-2 and 5-2b contained identical DNA sequences, with the
exception that clone 3-2 had a considerably longer 3' untranslated region,
5 including the poly (A) tail of the mRNA. The DNA sequence and predicted amino
acid seguence is shown in Figure 1g. The predicted amino acid sequence was
used to search the cGm6;. ,ed protein databases using the BLAST program
(Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J., 1990, J. Mol.Biol. 215, 403~10) on the National Centre for Biotechnology Information
10 computer, using ANGIS as an interface. The sequence was found to be highly
homologous with a 32kD lectin-like ~-galactoside-binding protein (GBP) from
Caenorhabditis elegans and Onchocerca volvulus. The O.circumcincta sequence
had a 69% identity with the C.elegans sequence and a 78% identity with the
O.volvulus sequence. The alignment of the amino acid sequences is shown in
1S Figure 1 h. By analogy with these homologous sequences, both the clones contain
the initiating ATG.
Proof that the doublet is a lectin-like ~-galactoside-bindin~ protein
Features that the doublet antigen and the C.ele~ns 32k GBP have in
common include size on SDS-PAGE, a lack of glycosylation and a blocked N-
20 terminus (see Hirabayashi, J., Satoh, M. and Kasai, K., 1992, J. Biol. Chem. ~I.15485-15490). A feature of the C.elegans GBP is that it can be affinity purified by
binding to an asialofetuin column (Hirabayashi, J., Satoh, M., Ohyama, Y. &
Kasai, K., 1992, J. Biochem. Tokyo 111, 553-555).
O.circumcincta L3 larvae were extracted in aqueous buffer (150mM NaCI,
25 2mM EDTA, 50mM tris-HCI, pH 8) and the extract was applied to an asialofetuin-
Affigel 15 column. Bound proteins were eluted with the above buffer containing
- 100mM lactose. Analysis by SDS-PAGE revealed that the doublet bound to the
asialofetuin column (Figure 1i). This demonstrates that the doublet is indeed a
lectin-like ~-g~l~ctoside-binding protein. As both bands of the doublet bind to the
30 sugar column, they are both lectin-like GBPs. It is not known why this GBP
appears as 2 bands in O.circumcincta as it is only a single band in C.elegans.
Perhaps the O.circumcincta protein undergoes some degree of post-translational
clea\/age.
K~,l~ ~1 (Rule 91)
WO95/09182 ~ ~ ~ 2 ~ ~ 2 PCT/AU94/00573
- 24 -
The function of these GBPs in nematodes is not known, but for C.elegans
it has been posh~l~ted that they are involved in regulation of morphogenesis, for
example in cuticle formation.
Fxpression of recomhinant ~ntigens
s Plasmid DNA from the clones or pMOSELOX control were transformed
into E~Qli BL21(DE3)pLysE cells using the simple transformation method of D.
Hanahan (In 'DNA Cloning: A Practical Approach', Volume 1, 1985 (ed. D.M.
Glover), IRL Press, Oxford, p115). It was necess~ry to transfer the plasmids from
BM25.8 to this strain because the recombinant antigens are expressed under
control of the T7 promoter, and BL21(DE3)pLysE cells carry the gene coding for
T7 polymerase under control of the ~ promoter. Colonies were picked and
grown overnight at 37C. 10mL cultures were inoculated at a 1:100 dilution from
the overnight cultures and grown for 5 hours. Recombinant protein synthesis was
induced by the addition of IPTG to 0.1mM and the incubation continued overnight.E.ÇQli cell pellets were resuspended in PBS containing 0.1% Triton X-100 and
disrupted by sonication. Insoluble material, including inclusion bodies, was
pelleted and resuspended by sonication in 1% CTAB. Samples were analysed by
SDS-PAGE and by Western blotting (Figures 1j and 1k). Clones 3-2 and 5-2b
produced fusion proteins which were positive on Western blots probed with the
anti-doublet mAb or with sheep antiserum to doublet antigen. In contrast, fusionproteins produced by clones 7-1, 7-2 and 8-2 were positive with the doublet mAb
but not with the sheep antiserum, confirming the results of the plaque
immunoassay described earlier (Figures 1b-1e). The pMOSELOX control protein
did not react with either antibody. All the recombinant fusion proteins, and thepMOSELOX control protein, were located exclusively in the CTAB-solubilised cell
pellet, indicating that they were expressed in inclusion bodies.
V~ccination trials
Three vaccination trials were performed using CTAB extracts of native Q
circumcincta antigens containing the 26-36kD immunoreactive region.
Vaccinated sheep were immunised 3 times, 2 to 3 weeks apart, with 50-
100~L9 of protein in quil A for each immunisation. Control sheep received quil Aonly. All immunisations were given intradermally. All sheep were challenged with
Rule 91)
~ WO 95/09182 ~ ~ 7 ~ 4 ~ 2 PCT/AUg4/00573
- 25 -
20,000 L3 larvae 2 to 3 weeks after the last immunisation and faecal egg counts
monitored. Results of three separate vaccination trials are shown in Figures
2a-2c and show clear reduction in faecal egg counts in the vaccinaled groups
compared to the co~ Jls.
s The first trial consial~d of 5 vac~,;.,dl~d and 3 control sheep. The second trial
had 10 \!acc;"dled and 8 control sheep. The third trial consisled of 7 vaccinates and 7
control sheep.
Species cross-reactivity
A 26-36kD antigen region was also identified in an O. ostertagii antigen
preparation when probed with sera from sheep vaccinated with O. circumcincta
26-36kD antigens (not shown). This indicates that similar antigens are also
present in this and possibly other nematode species.
Peripheral blood Iymphocyte proliferation assays of sheep at the end of
the third Osteri~.gia vaccination trial were peRormed using a crude soluble H.
contortus L3 extract. A highly significant (P~0.02) stimulation with the H.
contortus antigen was observed in the vaccinated sheep (mean 24003 cpm)
compared to the control sheep (mean 6122 cpm) indicating cross reactivity
between the two parasite species in the 26-36 kD antigen region. To ~ssess
crossprotection, all sheep of the third trial were drenched with ivermectin to
remove remaining Osterta~ia worms and i"rected with 10 000 H. contortus larvae.
As shown in Figure 2d, faecal egg counts were consistently lower in the
vaccinated compared to the control group suggesting that cross protection has
occurred. Rec~llse of the high variation in the faecal egg counts of the controlgroup, no sl~ lic~l significance was reached in the daily egg counts but there
was significant difference in the variance between the 2 groups (F-test). These
two results show that there is significant hetercl~gous stimulation and prote~;lion
between Haemonchus and Osterta~ia species and that similar protective
molecules are likely to exist.
Ch~ra~i~ri alion of 95-105 kn anti~en
Specific antisera prepared against the Haemonchus contortus 60-90 kD
surface antigen described in Australian Patent 640,364 also reacted with
O. circumcincta L3 larval extract in the same 95-105 kD region. This would
suggest that this is a similar antigen to that described for H. contortus.
Rule 9~)
WO95/09182 ~17 2 4 ~ 2 PCTIAU94100573 ~
- 26 -
FXAMPI F ?
TRICHOSTRONGYI US COI UBRIFORMIS
Fyrerimental Desi~n
Sheep were immunised by several i"rections with T. colubriformis and left
uninfected for at least 4 Illollllls. They were challenged with 50,000 I,
colub,irur,,,is L3 larvae, killed 10 days later and the first mesenteric Iymph node
(MLN) removed. Lymph node cells were processed and cultured as described for
O. circumcincta and the super"dl~"t was used to probe a Western blot of parasiteantigens. L3 larval antigen e)~l,dcts from O. circumcincta. H. contortus and I,
colubriformis were prepared as described previously for O. circumcincta.
The extracted antigens were run under reducing conditions on 12.5%
(w/v) SDS-PAGE and Western blotted onto PVDF membrane (Immobilon,
Millipore). The Western blot was probed with MLN-supernatant and developed
with a peroxidase conj!~gated anti-sheep Ig (DAKO).
Results and discussion
A strong reaction was observed with the T. colubriformis antigen extract
between the molecular weights of 32-35Kd and apparently consists of a doublet
(Figure 3). No or a weaker reaction was observed with the O. circumcincta and
H. contortus antigens. However, the T. colubriformis doublet antigen is in the
same position on SDS-PAGE as the O. circumcincta antigens described above
and it is therefore possible that both are essentially similar molecules but with
species-specific epitopes recognised most strongly by the hornologous
supernatant antibody probes.
FXAMpl F 3
FASCIOI ~ HFPATICA
We have used the technique of Western blotting with antibody probes
obtained as described in Australian Patent No. 640364 to identify putative
protective antigens of F. hepatica. A similar antigen may also be present in other
Fasciola species (eg. F. ~igantica) and in other trematode parasites (eg.
Schistosoma spp.).
Parasites and antigen extr~ctions
F. hepatica metacercariae (Mc) were obtained from Ciba-Geigy (N.S.W.,
Australia). Newly excysted juveniles (NEJ) were obtained by in vitro excystation
-- ~ = ~
WO 95/09182 ~7 PCTtAU94/00~73
as des~;,iLed previously (Au~l,alian Patent No. 640364). Juvenile liverflukes were
recovered from mouse livers 17 days after oral i"rtction. The different fluke
stages were sonicated in PBS co~,lai"i"g protease inhibitors, boiled in SDS non-reducing sample buffer, run on a 10% SDS-PAGE gel and l,ansfer,ed onto PVDF
s membrane (immobilon-P Mill" ore, MA) for Western blotting as described
previously (Patent No. 640364).
Preparation of culture super"dla,~t
In vitro cultures of spleen, I,epdlic Iymph node (HLN) and mesenteric
Iymph node (MLN) cells were set up at 3 x 106 cells per ml in culture medium
lo (DME co"lair,i"g 10% foetal calf serum, 2mM glutamine, 100u/ml penicillin, 100
~lg/ml sl,eplu,,,ycin and 2.5 x 10-5 2-",ercaptoell,anol) essentially as described
previously (Patent No. 640364). Supel"ala,~ were harvested after 4 to 5 days
incubation and stored at-20C until used.
F~perimental desi~n
8-9 week old PVC rats were infected with 100 F.11e~dlica Mc and treated
10 days later with the flukicide, Fasinex 120 (Ciba-Geigy) at 75 ~lg/gram. The
rats were challenged orally 6 weeks later with 200 Mc and killed 7 to 10 days after
challenge for collection of HLN, MLN and spleen cells.
Results
When no "breakthrough" infections had occurred (i.e. complete cure and
elimination of the primary infection by the flukicide) there was a distinct difference
in the local antibody response between the different Iymphoid organs after a
secondary challenge i"re~;tion. No reaction was observed when spleen or HLN
supernatants were used to probe a Western blot of NEJ antigen while a distinct
antigen doublet was recognised by supernatant from the MLN cells. This antigen
was located above the 110 kD molecul~ weight marker (Figure 4) and in later
experiments (not shown) was seen to I~ rale above a 200 kD molecular weight
marker and is further referled to as the ~200 kD a"li~en.
When signs of "breakthrough" infections were detected (i.e. Iiver
granulomas or adult flukes in bile ducts) a varied and complex pattern of NEJ
antigen recognition was observed with HLN supen,dldnl but the ~200 kD antigen
was again only and uniquely recognised by MLN cell supematant after secondary
WO 9S/09182 ~17 2 ~ ~ ~ 28 - PCT/AU94/00573
challenge infection (not shown).
All the rats were immune to the oral challenge i"re~ion as judged by the
absence of macroscopic~lly visible liver tracks and the total absence of juvenile
flukes from ",ashed whole liver prt:paldliGIls.
When the same MLN su~.er"ala.,l~ as above were reacted on a Western
blot with 17 day liverfluke antigen no such ~200 kD doublet reaction could be
observed (not shown) suggec;ling that this antigen is specific to the NEJ stage.Conclusion
A doublet antigen of ~200 kD molecular weight on a non-reducing SDS-
PAGE gel has been discovered which is present in the NEJ stage that is uniquely
recognised by culture su~,er~,~ld"l of MLN cells during an early secondary
response after oral cha"anye of immune rats. As the immunity developed in rats
against oral challenge i"rectiol) has been shown to occur at the level of the gut
(reviewed in Vet. rarasitol. 1986, ~Q, 63-93) this al,ligel) is a likely vaccinecandidate. The ~200 kD antigen appears to be stage specific for the NEJ fluke asit was not detected under similar conditions in flukes collected from liver tissue
and may therefore only be effective against the NEJ stage.
FXAMpl F 4
FASCIOI ~ HFPATICA
Parasites and antigen exlld1tiolls along with the preparation of culture
supernatant were carried out in the manner reported in Example 2.
F~perimental Desi~n
8-9 week old PVC rats were infected orally with 50 F. Hepatica Mc and
cured 14 days later with 150 ,~Lg Triclabend~ole~gram. They were given a
second oral infection of 150 Mc 3 days later and cured 4 days later as before. 1-2
months after the first infection rats were challenged orally with 400 IVlc and killed 7
days later for collection of HLN, MLN and spleen cells.
Monoclonal Antibody (mc~ Production
Rats were immunised and challenged as above and killed 5 days after the
challenge infection. MLN cell suspensions were p,~pared and fused with a Y3 rat
myloma line provided by the MRC cellular immunology unit, Oxford, United
Kingdom. Fusion supernatants were screened on Western blots against NEJ and
17 day liver stage antigen. Positive fusions were re-cloned out at least twice with
~ WO 95/09182 2~ 7 2 4 ~ ~ PCT/AU94/00573
-29-
rat thymocytes as feeders.
Surface Stailling with mc~hc
In vitro excysted NEJ's were incul7~t~d with mcAb supernatant containing
0.05% sodium azide for 30 minutes on ice. They were washed 3 times in cold
s PBS-azide and incl~h~te~ as before with a peroxid~-ce conjugated rabbit anti-rat
immunoglobulin (DAKO-Dnr"ark) diluted 1/20 in PBS. After 3 washes in PBS
colour dcvelopl"enl using Diamino benzidine sul,sllale was allowed to proceed
for a few minutes and stopped by dilution. After 2 more washes in PBS the NEJ
were fixed in PBS containing 1% fol",alclel,yde and 2% glucose and staining
10 evaluated under a light microscol)e.
Results
The ~200 kD antigen desc,iLed in the conclusion of Example 3 was also
present in the MLN supe",ala"l of rats immunised twice. In addition this hyper-
immune MLN super"aldl,t also recognised an antigen of apploxi",alely 32 kD and
a diffuse antigen(s) with app,u~ci",ate ~ ul ~r weight between 42 and 100 kD
(Figure 5). The 2 extra antigens were also only delect~cl on blots of NEJ antigen
and not on 17 day liverstage a"ligen.
Monoclonai Antibodies (m~A~
Several monoclonal antibodies were obtained from the fusion of twice
20 immunised and challenged MLN cells. Three of these mcAb's (F.h. 1-3) seemed
to recognise the 3 antigens detected with the MLN supen,ald"l~. One mcAb was
generated from the hepatic Iymph node of illre~;t~:d rats (FY1-6) and recognisedan antigen that was also strongly recognised with in~:dion serum on both NEJ
and liverstage fluke. All the mcAb's generated and their respective molecular
25 weight antigens are summarised in Table 1.
Later experiments (not shown) have established that the mcAbs FY4-7-
12 FY3-3-1 and FY3-3-2 still react with peritoneal fluke stages collected 2 daysbut not 4 days after infection of naive mice.
WO 95/09182 ~ PCT/AU94/00573
-30 -
T~RI F 1
Reactivity on
Western blots with
Antigen No. mcAb No. Approx. NEJ Liverstage
Molec~ r Weight Fluke
F.h. 1 FY4-7-12 ~200 ~ -
F.h. 2 FY3-3-1* 32 +
F.h. 3 FY3-3-2* 42-100 +
F.h. 4 FY3-5* 28 +
F.h. 5 FY4-7-6 37 + ND
F.h. 6 FY1-6* 54-55 + +
ND = Not done
* = Tested for surface staining on viable NEJ's
When reacted with viable NEJ's, 2 of the mcAb's (FY3-3-1 and FY3-3-2) reacted
5 with the surface of the NEJ fluke, each with a distinct staining pattern (Figure 6).
Rjochemical characterisation
The reduction sensitivity of the molecules recognised by the mcAbs was
examined by the addition of 2-mercaptoethanol (5% v/v final conc.) to the NEJ
protein extracts. There was a siy~ cant upward shift in the F.h.2 antigen after
lo reduction suggesting that this antigen contains a number of disulfide bonds. The
top bands recognised by mcAb FY3-3-2 migrated to a lower position on the gel
after reduction while there was no shift in mobility with the F.h.6 antigen.
To determine whether the mcAbs recognised carbohydrate epitopes,
SDS-PAGE gels of NEJ antigen extract were blotted onto PVDF membrane and
l 5 treated with periodate as described by Woodward, MP et al. (J. Immunol.
Methods, 78:143, 1985). After periodate treatment the reactivity of mcAbs FY3-3-2 and FY1-6 was abolished or drastically reduced respectively indicating that
these mcAbs react with carbohydrate epitopes. Periodate treatment of blots did
not change the reactivity of mcAbs FY3-3-1 and FY4-7. Recognition of a
20 carbohydrate epitope by mcAb FY3-3-2 explains its diffuse recognition pattern on
western blots as the carbohydrate epitope may be present on a range of
glycoproteins.
WO 95/09182 2 ~ 7 2 4 ~ 2 PCT/AU94/00573
-- 31 -
Amino acid sequence d~t~
The NEJ antigen preparations were run on SDS PAGE, blotted onto
ProBlot membrane (Applied Biosystems) and stained with Coomassie. Bands
corresponding to the position of antigens no. F.h.2 and F.h.6 were cut out and
5 directly sequenced on an ABI mould 476A protein sequencer. The N-terminal
sequences obtained were:
F.h.6: LEDNGRTHWAVLVA
F.h.2: KPNYKRQFEPFSDELIHYINLE
The sequences for F.h.2 shows 64.3% identity in 14aa overlap to Schistosoma
lo mansoni cathepsin B (Sm31) mRNA (Mol. Biochem. Parasitol. 33 : 113-122
(1 989)).
No clear homologies were found for F.h.6.
Conclusion
The present invention includes those antigens recognised by MLN
15 supernatant of immune challenged rats and all the antigens described in Table 1
and their respective antibodies. As the immunity developed in rats against oral
challenge infection has been shown to occur at the level of the gut and
peritoneum against the early fluke stages (reviewed in Vet. Parasitol. 1986, ~Q:63-93) these antigens are vaccine candidate. Four of the antigens appear to be
20 stage specific for the NEJ and 2 day old fluke as they were not detected under
similar conditions (Western blots) in flukes collected at later stages of infection
and are therefore likely to be involved in protection. Stage specific antigens have
not previously been identified in liver fluke and were only detected in the present
invention by using the novel antibody secreting cell probes.
Finally, it is to be understood that various other modifications and/or
alterations may be made without departing from the spirit of the present invention
as outlined herein.
