CA2147756A1 - Peptides and vaccines derived from nematode tubulin - Google Patents

Abstract

The present invention relates to a monoclonal antibody which substantially binds to .beta.-tubulin of nematode origin and frag-ments thereof. There is provided a hybridoma cell line producing the monoclonal antibody of the present invention which has been deposited at the ATCC under the accession number HB 11129. The antibody of the present invention can be used as an anti-parasitic agent and a diagnostic agent for parasitic diseases. The present invention also relates to the use of an antigen, which is recognized by the monoclonal antibody, as an immunizing agent and in vaccine compositions.

Description

WO94/10201 ~ 14 7 7 s 6 PCT/CA93/00431 PEPTIDES AND VACCINES DERIVED FROM NEMATODE TUBULIN

BACKGROUND OF THE INVENTION
l Field of the invention The present invention relates to a monoclonal antibody which speci~ically binds to ~-tubulin of nematode origin, which antibody can be used as an anti-parasitic agent and a diagnostic agent for parasitic diseases.
The present invention also relates to the use of immunogenic peptides useful in vaccine compositions ~or protecting mammals, such as humans and canines, against parasites of the Brugia and Dirofilaria genuses.
The present invention relates to the use of peptide fragments which provide improved means to protect a mammal against parasites of~ the Brusia and Dirofilaria genuses. More specifically, a dog exposed to the peptide fragments of the present invention is protected from heartworm by cytotoxic antibodies induced by the peptide.

Z. Brie~ descriPtion o~ Prior Art Farasitic diseases such as schistosomiasis (Bilharziasis) malaria and filariasis affect large numbers of people and are ~requent causes of gastrointestinal, circulatory and other disorders.
Parasitic infections often are chror.ic Gr recurreni, ~nd it is not surprising that immunologic types of diseases have been described.
Filariasis consists of a group of diseases occurring in tropical and subtropical countries and caused by Filarioidea.
Fil~riasis involves the lymphatic system, with obstruction leading to chyluria, hydrocoele, and elephantiasis that may involve the scrotum, legs and the arms.

WO94/10201 ~ 1~ 7 7 ~ ~ - 2-- PCT/CA93/00431 Etiology and Pathogenesis o~ Filariasis Wuchereria bancrof ti is ~ound only in humans; Brugia mal~yi is o~ten spread to man ~rom animal hosts. The adult filarial worms live in the human lymphatic system.
Micro~ilariae released by gravid ~emales are ~ound in the peripheral blood, usually at night. In~ection is spread by many species of mosquitoes. The microfilariae are ingested by the mosquito, undergo development in the insect's thoracic muscles, and, when mature, migrate to its mouthparts. When the infected mosquito bites a new host, the micro~ilariae penetrate the bite puncture and eventually reach the lymphatics, where they develop to the adult stage.

Pathology In~lammation and ~ibrosis occurring in the vicinity of the juvenile and adult worms produce pro-gressive lymphatic obstruction Symptoms and signs The incubation period may be as short as two mon~hs. The ~prelatent~ period, ~rom the time o in~ection to the appearance o micro~ilariae in the blood, is at least eight months. Clinica:L mani~estations depend on the severity of the infectioni they may include lymphan~itis, lymphadenitis, orchitis, funiculitis, epididymitis, lymph varices, and chyl-1ria. Chills, fever, headache, and malaise may also be present.
Elephantiasis and other late severe sequelae occur with l~ng-time residence in endemic areas and repeated reinfection. An aberrant form of filariasis (tropical eosinophilia) is characterized by hypereosinophilia, presence of microfilariae in the tissues but not in the blood, and high titers of anti~ilarial antibodies (tropical eosinophilia). Clinically, the patient may present with lymphadeno-splenomegly or with cough, bronchospasm, and chest infiltrates.

2 1 ~ 7 7 S ~ PCT/CAg3/00431 Diagnosis Microfilariae may be found in blood or lymph fluid. A number of serologic tests are available, but are not completely reliable. Antigen detection proce-dures are being investigated.

Tubulin Microtubules are proteinaceous organelles that are implicated in a variety o~ cellular functions including mitosis, intracellular transport, the maintenance of cell shape and the formation of cilia, flagella and sensory organelles. The major structural component of microtubules is tubulin, which is composed of a- and ~-subunits, the dimer having a molecular weight of llO kDa.
Both ~- and ~-tubulins are expressed as heterogeneous but closely related families of multiple isoforms, in different organisms, tissues and even within single cells of the same organism. The heterogeneous population of tubulin isoforms may result from both the differential expression of distinct tubulin genes and post-translational modifications. It has been suggested that the diversity in tubulin isoforms may have implications for specific MT functions (Lewis and Cowan, J. of Cell Biol., 1988, 106:2023-2033). The precise nature or role of a- and ~-tubulin isoforms have not yet been elucidated, although several groups of researchers have demonstrated that many in vivo functions of tubulin are to some extent, isoform specific (Gundersen et al., Cell, 1984, 38:779-789).
Benzimidazoles, anti-mitotic and anti-fungal agents are widely used in the chemotherapy o~ parasitic diseases. Several chemicals such as colchicine, vinblastine and benzimidazoles have been shown to bind to tubulin. Benzimidazoles exert toxic effects on nematodes by binding to tubulin and inhibiting polymerization of the heterodimer into microtubules. Benzimidazoles induce paralysis and slow growth in the free-living nematode Caenorhab~itis elegans. These drugs are potent ~ilaricides WO94/10201 21~ 7 7 5 6 PCT/CA93/00431 for B. pahangi and B. malayi. However, the precise benzimidazoles binding site has not been determined.
Monoclonal antibodies have made it possible to recognize dif~erent domains of tubulin in di~erent species in order to study the structure, distribution and ~unctions of tubulin. Tang and Prichard (Mol. & Biochem.
Parasitology, 1989, 32: 145-152) reported the presence of 4 to 5 a-tubulin isoforms in the tubulin-enriched extracts of adult B. pahangi. In addition, immunogold studies with ~. malayi adult and micro~ilariae using anti-tubulin monoclonal antibodies have revealed the presence o~ ~-tubulin in the somatic muscle blocks beneath the cuticle, intestinal ~rush border and intra-uterine micro~ilariae o~ the adult worms (Helm et al., Parasite Immunology, 1989, 11:479-502).
Several other anti-tubulin monoclonal antibodies raised against parasitic protozoa and nematodes have been isolated but these have been ~ound to cross-react with tubulin ~rom other ~pecies. For exampl~, Draber et al (Protoplasma, 1985, 128: 201-207) reported a monoclonal antibody raised against pig brain tubulin which reacted with microtubules ~rom dlverse species (mammalian, bird, amphibian, ~ungi, echinoderm, platyhelminth, slime moulds) but not protozoan tubulin. Similarly, Birkett et al. (FEBS Letters, 1985, 187: 211-218~ generated an anti-~-tubulin monoclonal antibody against P,~ysarum myxamoebae which reacts with ~-tubulin from various fungi, algae, higher plants, avian, insect and several mammalian sources. In addition, Helm et al. (Parasite Immunology, 1989, 11: 479-502) have raised monoclonal antibodies against microfilariae of Brugia species. Contrary to the anti-s. pahangi ~-tubulin monoclonal antibodies of the present invention, their monoclonal antibodies cross-reacted with mammalian tubulin.

All these monoclonal antibodies of the prior art are not specific against tubulin of nematode origin.
It would be highly desirable to have a monoclonal antibody which specifically binds to nematode tubulin and ~lA7756 WO94/10201 _ 5 _ PCT/CA93/00431 which could be used as an anti-parasitic agent and as a reliable diagnostic agent for parasitic diseases.
It would be also highly desirable to have a peptide which can be used to immunize m~mm~ls against parasites such as Brugia and Dirofilaria.
The desired peptide could be used in vaccine composition to provide an immune protection against these parasites.

SUMMAR~ OF THE INVENTION
In accordance with the present invention there is provided a monoclonal antibody which specifically binds to ~-tubulin of nematode origin and ~ragments thereo~.
The monoclonal antibody of the present invention can be used as an anti-parasitic agent and as a diagnostic agent for parasitic diseases.
In accordance with the present invention, there is also provided a hybridoma cell line which produces the monoclonal antibody of the present invention.
The monoclonal antibody of the present invention recognizes the C-terminal of nematode ~-tubulin which corresponds to a peptide of eighteen amino acids.
In accordance with another embodiment of the present invention, there is provided the use o~ a peptide as an immunizing agent against parasites wherein said peptide has the following amino acid sequence:
Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu, SEQ ID NO:l.
The use of the peptide in accordance with the present invention induces by a host the production of cytotoxic antibodies against parasites such as Brugia and Diro~ilaria.
In accordance with the present invention, there is also provided a vaccine for parasite in~ection comprising a peptide which has the following amino acid sequence:
Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu, SEQ ID NO:l 2147756 CA q3/~4~-~

Fin211y, in acco-dance with the ~resent invention, there is proYided a method cf immuniz~.ng m2mm21s againct parasites comprising the adminis-r2tion of t~e vaccine of th~
present invention. The ~-accine o the pr2sent invention c2n be adminis~ered in dosagD range o O C15 g to 1~ mg per kg bo~y weight, pref~rably in a dosage range of 1~ ~g to 0.15 mg per kg body weig~t.

BRIEF D~SCRIPTIO~a OF THE DRA-~7INGS
~ aving th~s generally desc~ibed the nature of ~he invention, re~erence will no+ 3e made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and where_r~
Fig. 1 is a Western blot analysis of extracts from par2si~es, pig br2in znd 3T3 fibroblasts using anti-B.pahangi ~ tubulin monoclonal antibody in Figure lA and anti-chick brain - tubulin monoclonal antibody in FiGure lB;
Fig. 2 is a Western blot analysis of the total protein extract of adult 3 . pahansi using anti-B. pahangi ~ tubulin monoclonal antibody P3D in Figure 2A or anti-B. pahangi ~ tu~ulin `t'~; ' monoclonal antibody lB6 in Figure 2B;
Pig. 3 is a Western blot analysis of products from limited proteolysis of anti-B. pahangi ~ tubulin monoclonal antibody P3D in ~igu~e 3A and anti-B.pahangi ~ tubulin monoclonzl antibody lB6 in Figure 3B;
Fig. 4 is a graph of the effects of anti-B. pahangi tubulin monoclonal antibody P3D on the via~ility of adult female B.phansi in vitro;
Fig. 5 is a graph of the effects of anti-B. pahangi tl~bulin monoclonzl antibody lB6 on the viability of adult femal2 B.phangi in vitro;
Fig. 6 is 2 graph of the e~fects of anti-chick br2in tubulin monoclonal anti~ody 357 on the viability of adult female B.phangi in vitro.

SUBSTITUTE SHE~T

W094/10201 ~14 7 7 5 6 PCT/CA93/00431 DET~TT.~D DESCRIPTIOh- OF THE lNv~:NlION
I-Monoclonal Antibody A first embodiment of the present invention relates to the production and characterization of a monoclonal anti-B.phangi tubulin monoclonal antibody.
The monoclonal antibody of the present invention, denoted P3D, specifically reacts to the C-t~rminAl portion of ~-tubulin from B. pahangi and Dirofilaria and SUB~ JTE SHEET

WO94/10201 ~1 4 7 7 5 6 7 _ PCT/CA93/0043l hence is capable of killing these parasites. The hybridoma P3D producing the monoclonal antibody of the present invention has been deposited at the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland, USA 20852) under accession number HB 11129 on September 18, 1992. This deposit is available to be public upon the grant o~ a patent to the a~signee, McGill University, disclosing same The deposit is also available as re~uired by Foreign Patent laws in countries wherein counterpart applications are filed.
In total, fifty-~our anti-s. pahangi tubulin mono-clonal antibodies were obtained after immunization of mice with purified B. pahangi tubulin. Because of their remarkable specificity for tubulin, monoclonal antibodies P3D and lB6 among others, have been selected for more extensive characterization. western blot analysis o~
one-dimensional SDS-PAGE showed that the anti-s. pahangi monoclonal antibodies o~ the present inven~ion recognized tubulin from a number of filarial nematode~ (B. pahangi, B
malayi and D. immitis ) and an ~ntestina' nematode ( H. conto~-tus). However, the monoclonal antibodies did not cross-react with tubulin ~rom pig brain, 3T3 mouse fibroblast cells or the parasitic protozoan G. muris. On the other hand, anti-chick monoclonal antibody 357 reacted with pig brain, 3T3 mouse fibroblast and G. murls tubulins as strongly as it did with filarial and other nematode ~-tubulins. The anti-s. pahangi tubulin monoclonal antibodies of the present invention recognize an epitope that is conserved between filarial and intestinal nematode ~-tubulin but not in protozoan and mammalian ~-tubulin. Whereas, cross-reactive anti-chick monoclonal antibody 357 recognizes an epitope that is conserved among filarial and intestinal nematodes as well as protozoan and mammalian ~-tubulin. The epitope recognized by monoclonal antibody 357 has been localized to a region of ~-tubulin between amino acid 339-417 in the proteolytic fragments of pig brain tubulin (Serrano et al., Analytical Biochemistry, 1986, 159: 253-259).
The anti-s. pahangi tubulin monoclonal antibodies of the WO94/10201 ~ 1~ 7 7 ~ 6 PCT/CA93/00431 present invention are highly specific to nematode tubulin.
The monoclonal antibodies o~ the present invention speci~lc ~or the ~- or ~-subunit o~ tubulin allow the subcellular localization and the ~unction o~ each subunit o~ tubulin to be studied. Proteins o~ the size o~
tubulin are generally built of several structural domains that have distinct functions. In the case of tubulin, such ~unctions include binding o~ anti-microtubule drugs, GTP or microtubule-associated proteins and the association between monomers, dimers or protofilaments.
The nematode-speci~ic anti-tubulin monoclonal antibodies of the present invention may serve to characterize the structure and distribution o~ B. pahangi tubulin molecule, and to de~ine microtubule stability and ~unctional domains.
The ~ollowing procedures are used in the prepa-ration and the characterization of the monoclonal antibody o~ the present invention.

Enzy~te-linked ;mm77nosorbent assay (ELISA) ELISA was per~ormed in microtite~ plates (Falcon) coated with the~ polylysine-purified tubulin or an 18 amino acid peptide corresponding to the extreme C-terminal residues 430-448 of B. pahangi tubulin (Guénette et al., Mol. and Biochem. Parasitology, l99l, 44: 153-164) at a concentration of l0~g/ml in phosphate bu~er saline (PBS). Plates are incubated wit~ 200 ~l of 1%
bovine serum albumin (BSA) in PBS. Horseradish peroxidase-labeled anti-mouse IgG or~ IgM (Bio-Can, Mississauga, Ontario) at dilutions of l:5000 and l:20,000, respectively, is added to eà~h well and incubated for l hour at 37C. The subskate is 2,21-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma).
The plates are read on a Titertek multiskan~ plate (Flow Laboratories, Irvine, Ayrshire, UK) at 414 nm. Normal mouse serum or culture medium used to ~row hybridoma cells ~Iscoves ntodified Dulbecco's mediu~(IMDM) with 20%
FCS, l0~ NCTC 135 and HT) is used as a n~gative control -Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Samples are run in a Mini Protean II~ dual slab cell (Bio-Rad, Richmond, CA) using 4% polyacrylamide as stacking and 12% polyacrylamide as separating gels Isoelectric focusing and two-~;men~ional electrophoresis (IEF-2D SDS-PAGE) IEF gels are prepared and run in tube gels (l.5 x 8cms) containing 9.5M urea tLKB) and 2~ (w/v) ampholines (LKB) (1.6% pH 4-6 and 0.4% pH 3.5-lO). IEF is conducted at 400 V or a period o~ 16 hours and then at 800 V ~or 3 h. Electrophoresis is per~ormed in 4% polyacrylamide stacking and 12% polyacrylamide separating gels, running at 50 V ~or 30 min and at 150 V ~or 60 min, in the Mini Protean II ~ slab cell A~ter 2-dimensional ( 2D ) SDS -PAGE, gels are either stained with silver stain (Bio-Rad), c~r the proteins are transferred ont~ nitrocellulose(NC) sheets for Western blot analysis.

Western blotting After l and 2D SDS-PAGE, tubulin subunits, individual tubulin iso~orms and peptides are elec-trophoretically transferred onto nitrocellulose sheets ~or 2 hours at 4C The nitrocellulose sheets are cut into several strips containing an identical pattern of separated proteins. To visualize protein bands, two nitrocellulose strips are stained with amido black. The remaining strips are washed in PBS and incubated for 2 hours at room temperature in 10% newborn calf serum (Gibco) in Tris-buffer saline (140 mM NaCl2, 50 mM Tris-HCl, pH 7.4, with O.l~ (v/v) Tween 20~ (TBS-T)) to saturate the unoccupied protein binding sites of the nitrocellulose. After washing, the strips are incubated overnight at 4C with anti-tubulin monoclonal antibodies (MAbs) or IMDM (negative control). The nitrocellulose strips are then washed 6 x 5 min with TBS-T, immersed in peroxidase-conjugated goat anti-mouse IgM or IgG (Bio-21~77Sfi Can) diluted at l:~00 with high salt buffer (l M NaCl2,lO mM Tris-HCl, pH 7.4; 0.5~ (v/v) Tween 20~ (HSB-T) with 10% NBCS)), and incubated ~or 2 hours at room temperature. After washing the nitrocellulose strips with TBS-T ~or 30 min, the bound peroxidase is detected with the substrate 4-chloro-l-naphthol (Sigma) at 3 mg/ml in methanol/PBS, 1:5 (vol/vol), containing 0.075~ o~ 30%
hydrogen peroxide.

l- Preparation of antigens Gerbils (Meriones unguiculatus), 9-lO months old and previously infected intraperitoneally with 400 B. pahangi infective larvae, are obtained from Dr. J. McCall (University o~ Georgia, USA). The adult B. pahangi (0.7 g) are harvested from the peritoneal cavities of gerbils in warm physiological saline (0.85% NaCl), washed with 0.025M bu~er containing l mM ethyleneglycol-bis-(~-aminoethyl ether)N,N,N',N'-tetraacetiC acid (EGTA), 0.5mM
MgSO4 and l mM guanosine-5'-triphosphate (GTP), and are homogenized in 7 ml of 2[N-morpholino)-ethanesulfonic acid (MES) buffer. The homogenate is centri~uged at lO0,000 g ~or l hour at 4C. The supernatant is retained and the pellet discarded. The same procedure is used to prepare tubulin ~rom other filarial ( B. malayi and D.
tis) and non-filarial nematodes (A. suum, benzimidazole-susceptible and resistant strains o~ H. contortus).
Tubulin rom pig brain is prepared by 2 cycles o~
polymerization-depolymerization.
Giardia Muris antigen is prepared as a sonicate.
A peptide corresponding to amino acid residues 430-448 of B. pahangi ,B-tubulin, synthesized using an Applied Bisystems Peptide Synthesizer~U, HPLC purified, sequenced and coupled to the carrier protein, keyhole Limpet Hemocyanin (KLH) (The Alberta Peptide Institute), is also used as an antigen in enzyme-linked im~unosorbent assay (ELISA).

W O 94/10201 21 i7 7 5 ~ - 11 - PC~r/CA93/00431 2- Puri~ication o~ parasite tubulin B. pahan~i, B. malayi, D. immltis, A. ~uum and ~. contortus tubulins are partially purified using polylysine a~inity chromatography (Lacey & Prichard, Mol. & Biochem. Para-sitology, 1986, 19: 171-181). The elution profile consisted of three distinct peaks. The first protein peak is eluted with MES buffer, the second with 1%
aqueous (NH4)zS04. Fractions for each peak are pooled and concentrated separately in centriflo TM ( Amicon) at 400 g-Polylysine-puri~ied proteins are separated on SDS-PAGE, protein bands of the molecular weight corresponding to tubulin are excised, and the protein is electro-eluted (Electroeluter~M, Bio-Rad) (Blose et al., J. of Cell Biol. , 1984, 98: 847-858). The eluted protein is precipitated three times with 80~ acetone at -20OC or 5 hours and then dissolved in 0.125 M Tris--HCl (pH 6 8), 0 1% SDS and 1 mM EDTA, dialysed overnight against this buffer at 4C
and stored at - 70C until used Crude supernatant o~ adult B. pahangi iS chro-matographed on a polylysine agarose column The protein content of each ~raction is determined The elution profile consisted of 3 distinct protein peaks The protein concentrations in the first and second peaks are very high compared with that in the third peak, but in contrast to this last peak the first two peaks contained little if any tubulin This is consistent with the previous report by Tang & Prichard (Mol & Biochem Parasitology, 1989, 3Z: 145-152) Third peak proteins are concentrated and then subjected to SDS-PAGE, respectively The tubulin band is cut Otlt of the SDS-gels and subjected to electro-elution for further puri~ication 3- Tmm~ln;zation and preparation of monoclonal anti-bodies Six week old female BALB/c mice (Charles River Canada Inc , St Constant, Quebec) are injected subcu-taneously at three week intervals with purified eluted B.

21477~6 W O 94/10201 - 12 - PC~r/CA93/00431 pahangi tubulin (100 ~g/injection) using equal volumes of complete Freund's adjuvant for the first injection and incomplete adjuvant for the second injection. The third immunization o~ 100 ~g of tubulin in PBS is administered intraperitoneally (i.p.). At this stage, mice are bled and serum is tested for anti-tubulin antibodies by ELISA
and Western blotting. The spleen cells from the mouse giving the highest titer are fused with the myeloma cell line, P3X63.Ag8 (American Type Culture Collection (ATCC), accession number CRL1580, Rockville, MD), as described by Hurrell ("Monoclonal hybridoma antibodies: Techniques and applications", 1982, CRC Press, Boca Raton, Florida, p.
22). Positive cultures as determined by ELISA and Western blotting, are cloned twice by limiting dilution.
Two different isotypes of anti-s. pahangi mono-clonal antibodies were obtained. Seven out of fifty-four monoclonal antibodies were polyreactive IgM, recognizing tubulin as well as other high and low molecular weight proteins, whereas the remaining monoclonal antibodies represented two populations of the IgG isotype. Of these, four out of fifty-four reacted with tubulin and other low molecular weight proteins; however, ~orty-three monoclonal antibodies were specific for tubulin.
Monoclonal antibodies P3D and lB6 specific to nematode tubulin, were chosen for further characterization. These monoclonal antibodies are of IgG isotype.

4- Monoclonal antibodies (MAbs) Three monoclonal antibodies, all specific for tubulin, are investigated. Anti-chick brain monoclonal antibody 357, which cross-reacts with ~-tubulins from a spectrum of eukaryotic cell types, was purchased from the Radiochemical Centre (Amersham, England) and monoclonal antibodies P3D and lB6 are raised against the tubulin of adult s. pahangi. All anti-tubulin monoclonal antibodies are of IgG isotype.

WO94/10201 ~14 7 7 5 ~ 13 - PCT/CA93/00431 5- Specificity o~ monoclonal antibodies (MAbs) P3D, 1~6 and 357 The specificity of these monoclonal antibodies is investigated by determining their reactivity to proteins rom a variety of ~ilarial and non-filarial nematodes, protozoa and m~mmAlian cells using ELISA and Western blot.
In ELISA, the anti-~. pahangi monoclonal antibodies P3D and lB6 do not react with G. muris tubulin, which is recognized by anti-chick brain tubulin monoclonal antibody 357. Crude and partially ,purified extracts of adults and micro~ilariae o~ s. pahangi, adult s malayi and D.
i~runitis, eggs of H. contortus, adult A. suum, pig brain and 3T3 mouse fibroblast cell tubulins are separated on SDS-PAGE and electrophoretically trans~erred onto nitrocellulose sheets The blots are treated with: (l) amido ~lack; ( 2 ) monoclonal antibody lB6 i ( 3 ) monoclonal antibody P3D; and monoclonal antibody 357. Analysis of amido black stained blots revealed that crude extracts of adults and microilariae o B. pah~ngi, adult B. malayl and D. immitis, eggs o~ susceptible and resistant strains o~ ~.
contortus, adult A. suum, pig brain and 3T3 mouse ~ibroblast cell contained many bands in the tubulin region. Tubulin from the various nematodes and mAmm~lian extracts are separated into two bands designated a and ~. Anti-s.
pahangi monoclonal antibody P3D recognized specifically ~-tubulin from adult and microfilariae of the filarial worms s. pahangi, B malayi and D. immitis (Fig. lA, lane 1-4).
It also reacted with equal intensity to tubulin from the intestinal nematode H. contortus ( BZ-susceptible and benz-imidazole-resistant strains) (Fig. lA, lane 5-6).
Tubulin from Æ. suum do not show very strong reactivity with this monoclonal antibody (Fig. l~, lane 7), no reactivity to 3T3 mouse fibroblast cells or pig brain tubulins is detected (Fig. lA, lane 8-9). Similar results are obtained using monoclonal antibody lB6 (not shown). Whereas, cross-reactive anti--chick ~-tubulin monoclonal antibody 357 recognized ~-tubulin from all nematodes and m~mm~ lian cells (Fig. lB, lane l to~9).

WO94/10201 ~1 4 7 7 5 6 PCT/CA93/00431 6- Identification Of tubulin isof o~ms Anti-s. pahangi ~-tubulin monoclonal antibodies P3D
and ls6, and anti-chick ~-tubulin monoclonal antibody 357, are used to characterize ~-tubulin isoforms in B.
pahangi tubulin. Monoclonal antibodies P3D (Fig. 2A) and 357 recognized the same isoform pattern, reacting with two ~-tubulin isoforms in the crude as well as partially purified extracts of B. pahangi ( not shown). Whereas, monoclonal antibody lB6 specifically recognized only one ~-tubulin isoform in the extract of B. pahangi ( Fig. 2B).
The ~-tubulin isoforms are in the pH range o~ 5.1-5.3.
Monoclonal antibody 357 probed blots are re-probed with monoclonal antibodies P3D and lB6 respectively, to demonstrate that the same spots are recognized by this monoclonal an~ibody. Furthermore, to show the ~ull complement o~ ~-tubulin iso~orms, P3D and lB6 probed blots are re-probed with monoclonal antibody 357. The results indicated that all these monoclonal antibodies recognized the same isoforms in tubulin-enriched extracts of adult B. pahangi. However, monoclonal antibody lB6 is speci~ic to one isoform 7- Limited Proteolysis o tubulin Limited proteolysis o~ tubulin in gel slices is per~ormed. Gel pieces corresponding to the tubulin are cut out of the polyacrylamide gels and placed directly into the sample well of a second 15% SDS-polyacrylamide gel. Gel pieces are overlaid with one of the following proteases:~-chymotrypsin from bovine pancreas (Sigma) or 5. aureus V8 protease (Boehringer Mannheim). The SDS-PAGE
is performed at 50 V until bromophenol blue dye reached the bottom of the stacking gel and then increased to 150 V for the remainder of the electrophoresis. After SDS-PAGE, the digested peptides are either stained with silver stain or transferred onto nitrocellulose sheets, in the same way as described for the Western blot analysis, and reacted either with anti-B. pahangi tubulin WO94/10201 214 7 7 ~ - 15 - PCT/CA93/00431 monoclonal antibodies or anti-chick tubulin monoclonal antibody 357.

8- Interaction o~ anti-tubulin monoclonal sntibodieæ
with tubulin proteolytic fragments Three identical gels are run and the peptide fragments transferred onto nitrocellulose, Three o~ which are immunostained with anti-s. pahangi tubulin monoclonal antibodies P3D and lB6 (Fig. 3) and anti-chick brain ~-tubulin monoclonal antibody 357 (not shown).
Western blots of peptides digested with chy-motrypsin showed that monoclonal antibody P3D reacted with a Zl kDa chymotrypsin fragment (Fig. 3A, lane 2) and a 21 kDa v8 protease ~-tubulin ~ragment (Fig. 3A, lane 3). In contrast, monoclonal antibody lB6 reacted with the two chymotrypsin-digested ~ragments o~ 42 and 34 kDa ( Fig . 3B, lane 2). It reacted strongly with the 42 kDa and weakly with the 34 kDa ~ragment. However, the same protease (Fig. 3B, lane 3). These results of the limited proteolysis analysis lndlcate that the antigenic site recognized by monoclonal antibody P3D differs from that recognized by monoclonal antibody lB6.
Although ~monoclonal antibody 357 reacts strongly to intact ~-tubulin from B, pahangi, no interaction was seen with ~-tubulin fragments digested w:ith chymotrypsin or V8 protease (not shown). Protease digestion appears to destroy the reactivity of B. pahangi tubulin towards monoclonal antibody 357.

II-Peptide used as an ;r~llnizin~ a~ent a~c~inst ~arasite The monoclonal antibody P3D of the present inven-tion recognizes the C-terminal of nematode ~-tubulin which corresponds to a peptide of eighteen amino acids.
A second embodiment of the present invention relates to the use of a peptide, recognized by the antibody of the present invention, which consists of the following eighteen amino acid sequence:

21477~ =.i;

Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln1 5 10 15 Glu Glu, SEQ ID NO:l, which is located at the C-terminal of nematode ~-tubulin. Furthermore, the present invention relates to a vaccine which comprises a peptide that consists of the following amino acid sequence:
- Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu, SEQ ID NO:l.
The present invention provides a peptide having the amino acid sequence derived from the eighteen amino acids at the C-terminal of ,B-tubulin from B. pahangi and Dirofilaria. The peptide can be made using a peptide sequence or using recombinant DNA technology.
A vaccine comprising the peptide of the present invention, a fragment thereo~ or a larger peptide which comprises the amino acid sequence of the peptide of the present invention is effective in conferring protection against parasite infection. Such vaccines can be prepared by one having ordinary skill in the art.
It has been discovered that monoclonal antibodies which speciically react to the C-terminal portion of ~-tubulin from B. pallangi or Dirofilaria are capable o~ killing these parasites.
Accordingly, using a vaccine that comprises peptide with the epitope of the C-terminal of B. pahangi or Dirofilaria ,~-tubulin will elicit cytotoxic antibodies in vaccinated m;3mm~ls that can kill these parasites and therefore protect the mammal against the parasite~
The present invention relates to vaccines which comprise a peptide which consist of the eighteen amino acid residues from the C terminus of B. pahangi or Dirofilaria ,~-tubulin or fragment thereof and to vaccines which comprise a peptide that have portions which are the eighteen amino acid sequence.
The amino acids at the carboxy terminus of Brugia and Dirofilaria ,~-tubulin are:

2~ ~7~6 DEEGDLQEGESEYIEQEE or Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu, SEQ ID NO:1, or aspartate-glutamate-glutamate-glycine-aspartate-leucine-glutamine-glutamate-glycine-glutamate-serine-glutamate-tyrosine-isoleucine-glutamate-glutamine-glutamate-glutamate.
Production of the peptide of the present invention, ~ragment thereo~ or larger peptides which include this sequence can be accomplished using standard peptide synthesis or recombinant DNA techniques both well known to those having ordinary skill in the art. Peptide synthesis is the preferred method of making polypeptides which comprise about 50 amino acids or less. For larger molecules, production in host cells using recombinant DNA
technology is pre~erred Smaller peptides according to the present invention can be synthesized, for example, by solid-phase methodology utlllzlng an Applied Bi~y~t~m~ 43 OA p~ptide synthesizer (Applied Biosystems, Foster City, California) as described in detail below For larg~er molecules, production in host cells usin~ recombinant DNA is preferred. There are several different methods available to one having ordinary skill in the art who wishes to use recombinant DNA technology to produce proteins. Typically, genes encoding desired polypeptides are inserted in expression vectors which are then used to transform or transfect suitable host cells.
The inserted gene is then expressed in the host cell and the desired polypeptide is produced.
Methods and materials for preparing genes and recombinant vectors, transforming or transfecting host cells using the same, replicating the vectors in host cells and expressing biologically active foreign peptides and proteins are described in Principles of Gene Manipulation, by Old and Primrose, 2nd edition, 1981 and Sambrook et al., Molecular Cloning, 2nd Edition, Cold WO94/10201 ~1 4 7 7 5 6 PCT/CA93/00431 Spring Harbor Laboratory Press, NY (1989), the disclosure of both is incorporated herein by re~erence.
European Patent application 322,Z37 published on June 28, 1989, U.S. Patent 4,735,801 (Stocker, April 5, 1988) and U.S. Patent 4,837,151 (Stocker, June 6, 1989) describe attenuated microorganisms use~ul in vaccine and which microorganisms have non-reverting mutation in two discrete genes in their aromatic biosynthetic pathway.
These microorganisms can usefully form the basis of an oral live vaccine and can be genetically engineered so as to express antigens from other pathogens. These references are all incorporated herein by re~erence.

ExamPle I
I~ v5tro assay of B. r~h~ inhibition Measurement o~ the in vitro activity o~ anti-s.
pahangi tubulin monoclonal antibodies P3D, lB6 and anti-chick brain tubulin monoclonal antibody 357 against emale s. pahangi or anti-chick tubulin monoclonal anti-bodies can independently cause any damage to the intact adult worms. Mebendazole (MBZ) is used to determine whether the presence o~ MBZ drug alone or in synergy with monoclonal antibodies has any di~erential efect.

InhibitQrs Anti-s. pahangi ~-tubulin monoclonal antibodies P3D, lB6 (in culture medium)), anti-chick ~-tubulin monoclonal antibody 357 (in ascites fluid) and mebendazole (MBZ) (in DMSO), a benzimidazole anthelmintic drug, are used as inhibitors in the in vitro assays. Anti-s. pahangi anti-chick brain monoclonal antibody 357 is in ascites fluid and is diluted to 1:1000 concentration with culture medium IMDM/FCS.

Culture in ~itro Parasitic nematodes are isolated from their mammalian host. B. pahangi are isolated from peritoneal cavities of gerbils, as described earlier in a sterile hood of Iscove's Modified Dulbecco's Medium/NCTC-135 WO94/10201 ~ 7 7 ~ l5 - PCT/CA93/00431 supplemented with 20% fetal calf serum (IMDM/FCS).
Following isolation, B. pahangi are washed ~ive times with sterile IMDM/FCS medium, for sur~ace sterilization.
Three wells in 24-well plates (Nunc) are set up ~or each test monoclonal antibody, drug and ~or the control cultures. To each well was added 2 ml o~ t:he appropriate test medium containing pure monoclonal antibody P3D, lB6, 357 alone or monoclonal antibody and MBZ and two adult worms. The plates are incubated at 37C in a humidi~ied incubator in the presence of 95% air and 5% CO2. Worm activity is observed every two hours, and motility is assessed subjectively by observation with a naked eye.
Experiment is terminated ater 48 hours. During the 48 hour incubation the culture medium is not changed.
Control medium contained an identical volume o~ the IMDM/FCS without monoclonal antibodies or drug.

Optimization of MTT reduction assay Female live B. pahangi worm iS place~ in 0.5 ml of IMDM containing 0.5 mg/ml [3-(4,5-dimethyl~thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma) (MTT) and incubated at 37C for various time interva]s ranging from 0-90 min (MTT-~eduction). Female worms that had previously been heat-killed are also incubated with MTT
for selected time intervals over this range. For each time point three replicate worms are used. At the end of the MTT incubations worms are removed and careully transferred to a separate well of a microtiter plate containing 200~1 of DMSO and allowed to stand at room temperature for l hour (formazan solubilization), with occasional gentle agitation to evenly disperse the color.
The absorbance of the resulting formazan solution is then determined at 550 nm, using an ELISA reader and compared with a DMSO blank.

Quantification of B . p~h~n~ viability A three-step colorimetric assay based on MTT is used to assess viability of parasitic nematodes. MTT is dissolved in PBS at a concentration of 5 0 mg/ml and sub-~14~7~6 sequently diluted to 0.5 mg/ml with PBS. Worms are incu-bated for 30 min at 37C (MTT reduction). A~ter incu-bation, worms are transferred to 96 well plates con-taining 200~1 of DMSO. The plates are allowed to stand for l hour at room temperature (formazan solubilization).
The absorbance is determined at 550 nm in the presence and absence of worm and compared with a DMSO blank.
Worms are killed for control purposes by heating in PBS
at lO0C for lO min.
Previous studies have demonstrated the utility of MTT-formazan colorimetry in proliferation and cytotoxicity assays in anti-cancer chemotherapy. Sub-sequently it has been demonstrated that the application of this assay was success~ul to determine filarial viability and ~or in vitro anti-filarial drug screening.
MTT is pale-yellow in solution but when incubated with living cells is reduced by active mitochondria to yield a dark blue crystalline deposit (formazan) within cells, which once solubilized can be quantified colorimetrically.
In accordance with the present invention, MTT
assays are per~ormed to determine the effects of anti-tubulin monoclonal antibodies on the viability of parasitic nematodes. Viable control female s. pahangi showed rates of formazan formation that are maximal and linear during the irst 30 min o~ the incubation with MTT. By one hour rate of formazan formation had begun to decline and plateaued between 60-90 min. Heat-killed worms shows only background levels of formazan formation.
Worms treated with anti-s. pahangi monoclonal antibody P3D alone and in synergy with MBZ show a detectable decrease in motility 12 hours post-treatment.
- The other anti-s. pahangi monoclonal antibody lB6 alone and in synergy with MBZ, also exhibit an apparent decline in the motility of worms, however, no mortalities are observed using these monoclonal antibodies during the experiment. No noticeable reduction is observed in the motility of the worms treated with MBZ alone or anti-chick brain monoclonal antibody 357 alone or in synergy WO94/10201 21~ 7 7 5 6 ~1 PCT/CA93/00431 with MBZ or the control worms, during the period the worms are in culture. From these obser~ations, it is suggested that the reduction in the worm motility is caused mainly by the anti-B~ pahangi monoclonal antibody alone, since MsZ alone do not have any e~ect on the motility o~ the worms.
Analysis of MTT assays demonstrates that mono-clonal antibody P3D treated B. pahangi shows significant decline in their ability to reduce MTT to formazan (Fig.
4). This monoclonal antibody alone caused a highly signi~icant 80% reduction in worm viability, compared with untreated live worms, 48 hours post-treatment. MBZ
in synergy with monoclonal antibody P3D caused significant 70g reduction in the viability of worms. The high reduction in the viability o~ worms seems to be due to the presence o~ monoclonal antibody P3D and not MBZ
As MBZ alone induced a minimal decrease (lO~) in the viability of worms. Exposure to monoclonal antibody lB6 resulted in 40% decrease in the ability of worms to reduce MTT (Fig. 5). Monoclonal antibodies P3D and lB6 had the same respective e~ects on the viability o~ males as for females. Anti-chick l~-tubulin monoclonal antibody 357 did not show any significant effect on the viability o~ worms (Fig. 6). The properties of anti-s. pahangi and anti-chick brain monoclonal antibodies appear qualitatively similar. Differences in their inhibitory effects on the motility and viability of 3. pahangi may depend on their different binding affinities.
Control untreated live female worms show a linear rate of formazan production and gave an absorbance reading of l.l at 550 nm. In contrast, heat killed worms show no ability to reduce MTT (~ig. 4 to ~). After DMSO
solubilization for l hour the absorbance of the resulting formazan solution is determined in the presence or absence of the female worms. This is done to determine if the presence of worm had any effect on the absorbance values. In the presence of worm, there is a slight increase in the absorbance values obtained. Inhibition of MTT reduction does not always occur ~lniformly along ~147756 22 - !~ i the entire length o~ treated worms and areas retaining viability are observed. Thus by close observation of the worms during MTT reduction it is sometimes possible to - determine sites of selective damage.
Conclusion The results o~ Example I demonstrate an apparent decline in the motility, when the worms are cultured with the anti-tubulin monoclonal antibodies P3D and lB6 of the present invention. However, no noticeable reduction in the motility is observed, when the worms are treated with anti-chick monoclonal antibody 357, MBZ or IMDM/FCS
culture medium without antibodies. The viability of the worms was assessed by MTT assay. The anti-s. pahangi, monoclonal antibodies P3D and lB6 o~ the present invention, signi~icantly reduced the viability o~
parasitic nematodes. No reduction in viability was observed when adult B, pahangi were exposed to anti-chick monoclonal antibody 357 and/or MBZ.

~.Y~m~le II
Anti-parasitic Antibody composition An antibody composition to be administered to a gerbil as an anti-parasitic agent in dosage varying ~rom lmg/0.5ml to lOmg/0.5ml in a pharmaceutical carrier suitable ~or intraperitoneally administration.
The carrier for such administration is an IMDM
culture media.

~Y~m~le III
Production o~ the eighteen Am-ino Acid peptide The peptide consists of the amino acid sequence:
Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu, SEQ ID NO:l.
To prepare the peptide for use in a vaccine, the peptide is synthesized by solid phase methodology on an Applied I - - r .; ~

Biosystems Inc (ABI) 430A peptide syn~hesizer using ABI's Small Scale Rapid Cycles (SSRC) o~ a 0.1 mmole scale or other similar synthesizer. SSRC utilizes abbreviated single couple cycles with standard Boc chemistry. The t-Boc-L-amino acids used (1 mmole) are supplied by ABI with standard side-chain protecting groups. The completed peptide is removed from the supporting PAM (phenylacetamidomethyl) resin, concur-rently with the side-chain protecting groups, by a standard HF procedure using appropriate ca~ion scavengers (10% v/v amisole, p-cresol plus p-thiocresol, 1,4-butanedithiol plus anisole or DMS plus anisole) depending on the amino acid sequence o~ the peptide.
The crude peptide, a~ter HF cleavage, is purified by preparative reverse phase chromatography on a Phenomenex C - 18 Column (250 x z2.5 mm) using water acetonitrile gradients, each phase containinq 0 1% TFA.
The pure fractions (as determined by analytical HPLC) are pooled, acetonitrile evaporated and the aqueous solution lyophilized. The peptide is analyzed by ~ast atom bombardment mass spectrocopy and resulting ( M+H ) + is compared with the anticipated (M+H)+

Exam~le IV
Vaccine Comprising Eighteen Am; no Acid Peptide The peptide can be prepared in vao~cine dose ~orm by well-known procedures The vac:cine can be administered sublingually, intramuscularly, subcuta-neously or intranasally. For parenteral administration, such as intramuscular injection, the immunogen may be combined with a suitable carrier, for example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunomodulating agents such as aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dlpeptide, bacterial endotoxin, lipid X, Corynebacterium parum (Propionobac~erium acnes), Bordetella pertussis, polyribonucleotides, sodium -2~477S~

alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants-. Such adjuvants are available commercially ~rom various sources, for example, Merck Adjuvant 65 (Merck and Co., Inc., Rahway, NJ).
The proportion o~ immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount o~ about 0.5% of the vaccine mixture (Al2O3 basis). On a per dose basis, the concentration of the immunogen can range from about 0.015 ~g to about l.5 mg per kilogram per patient body weight for an animal or human patient. A pre~erable dosage range in humans is about 0.l - l ml, preferably about 0.l - ml. Accordingly, a dose ~or intramuscular injection, ~or example, would comprise 0.l ml containing immunogen in admixture with 0.5% aluminum hydroxide.
The vaccine of the present invention may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments such as antibiotics.
While the invention has been described in con-nection with speci~ic embodiments thereof, it will be understood that it is capable of further modification and this application is intended to cover any variations, uses, or adaptations o~ the invention ~ollowing, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows from the scope of the appended claims.

WO94/10201 21~ 7 7 5 ~ -25- PCT/CA93/00431 ~

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: McGill University 845 Sherbrooke Street Nest Montreal, Quebec, ~ANAnA H3A. lBl (ii) TITLE OF l~v~N'l'ION: Peptides and Vaccines Derived From Nematode Tubulin (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MERCHANT & GOULD
(B) STREET: 3100 Norwest Center (C) CITY: M; n~eApolis (D) STATE: MN
(E) COUNTRY: USA
(F) ZIP: 55402 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk ( B ) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC--DOS/MS--DOS
(D) SOFTWARE: PatentIn Release #1.0, ~ersion #1.25 (vi) CURRENT APPLICATION DATA:
~ A ) APPLICA~ION NUMBER !
(B) FILING DATE:
(C) CLASSIFICATION
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/967,829 (B) FILING DATE: 28-OCT-1992 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Woessner, Warren D.
(B) REGISTRATION NUMBER: 30,440 (C) REFERENCE/DOCKET NUMBER: 10022.3-WO01 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 612-332-5300 (B) TELEFAX: 612-332-9081 ~ 21~7756 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu

Claims (12)

WE CLAIM

1. A peptide having the amino acid sequence:
Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser Glu Tyr Ile Glu Gln Glu Glu (SEQ I-D NO:1), or a fragment thereof for use as a vaccine for protecting against filarial parasite infection.

2. A compound having at least the amino acid sequence:
Asp Glu Glu Gly Asp Leu Gln Glu Gly Glu Ser . Glu Tyr Ile Glu Gln Glu Glu (SEQ ID NO:1), for use as a vaccine for protecting against filarial parasite infection.

3. A peptide accordlng to claim 1 or 2, wherein the filarial parasite is Dirofilaria immitus.

4. A peptide according to claim 1 or 2, wherein the parasite is Wuchereria bancrofti.

5. A peptide according to claim 1 or 2, wherein the parasite is Brugia malayi .

6. A peptide according to claim 1 or 2 that reduces the worm burden.

7. A peptide according to claim 1 or 2 that reduces the microfilarial load.

8. Use of a peptide according to claim 1 or 2 for preparing a vaccine composition for protecting against filarial parasite infection.

9. Use of a peptide according to claim 8 to prepare a vaccine for protecting against Dirofilaria immitus.

10. Use of a peptide according to claim 8 to prepare a vaccine composition that has a dosage range of 0.015 µg to 0.75 µg per kg body weight.

11. Use of a peptide according to claim 8 to prepare a vaccine composition for protecting against Brugia malayi.

12 Use of a peptide according to claim 8 to prepare a vaccine composition for protecting against Wuchereria bancrofti.