EP0674659A1 - Nematodentubulin abgeleitete peptide und impfstoffe - Google Patents

Nematodentubulin abgeleitete peptide und impfstoffe

Info

Publication number
EP0674659A1
EP0674659A1 EP93922866A EP93922866A EP0674659A1 EP 0674659 A1 EP0674659 A1 EP 0674659A1 EP 93922866 A EP93922866 A EP 93922866A EP 93922866 A EP93922866 A EP 93922866A EP 0674659 A1 EP0674659 A1 EP 0674659A1
Authority
EP
European Patent Office
Prior art keywords
glu
tubulin
peptide
monoclonal antibody
gin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93922866A
Other languages
English (en)
French (fr)
Inventor
Roger K. Prichard
Nasreen Inayat Bughio
Gaetan Mario Faubert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
McGill University
Original Assignee
McGill University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by McGill University filed Critical McGill University
Publication of EP0674659A1 publication Critical patent/EP0674659A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a monoclonal antibody which specifically 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 for protecting mammals, such as humans and canines, againsr parasites of the Br ⁇ gia and D ⁇ rofiiaria genuses .
  • the present invention relates to the use of peptide fragments which provide improved means to protect a mammal against parasites of the Brug ⁇ a and D ⁇ r-f ⁇ la - ⁇ a 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.
  • Parasitic diseases such as schistoscmiasis (Bilharziasis ) malaria and filariasis affect large numbers of people and are frequent causes of gastrointestinal, circulatory and other disorders. Parasitic infections often are chronic or recurrent, and 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 Filario ⁇ dea .
  • Filariasis involves the lymphatic system, with obstruction leading to chyluria, hydrocoele, and elephantiasis that may involve the scrotum, legs and the arms .
  • Etiology and Pathogenesis of Filariasis involves the lymphatic system, with obstruction leading to chyluria, hydrocoele, and elephantiasis that may involve the scrotum, legs and the arms .
  • W chereria bancrofti is found only in humans ; Brugia malay ⁇ is often spread to man from animal hosts .
  • the adult filarial worms live in the human lymphatic system.
  • Microfilariae released by gravid females are found in the peripheral blood, usually at night. Infection 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 outhparts .
  • the infected mosquito bites a new host the microfilariae penetrate the bite puncture and eventually reach the lymphatics, where they develop to the adult stage.
  • the incubation period may be as short as two months.
  • the "prelatent” period from the time of infection to the appearance of microfilariae in the blood, is at least eight months.
  • Clinical manifestations depend on the severity of the infection; they may include lymphangitis, lymphadenitis, orchitis, funiculitis, epididymitis, lymph varices, and chyluria. Chills, fever, headache, and malaise may also be present.
  • Elephantiasis and other late severe sequelae occur with long-time residence in endemic areas and repeated reinfection.
  • An aberrant form of filariasis is characterized by hypereosinophilia, presence of microfilariae in the tissues but not in the blood, and high titers of antifilarial antibodies (tropical eosinophilia).
  • the patient may present with lymphadeno-splenomegly or with cough, bronchospasm, and chest infiltrates.
  • 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.
  • Microtubules are proteinaceous organelles that are implicated in a variety of 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 ⁇ - and ⁇ - subunits, the dimer having a molecular weight of 110 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.
  • 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 ⁇ - 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 of 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 Caenoxhabditis elegans .
  • These drugs are potent filaricides for B. pahangi and B. malay .
  • the precise benzimidazoles binding site has not been determined.
  • the desired peptide could be used in vaccine composition to provide an immune protection against these parasites.
  • a monoclonal antibody which specifically binds to /?-tubulin of nematode origin and fragments thereof.
  • the monoclonal antibody of the present invention can be used as an anti-parasitic agent and as a diagnostic agent for parasitic diseases.
  • the monoclonal antibody of the present invention recognizes the C-terminal of nematode 7-tubulin which corresponds to a peptide of eighteen amino acids.
  • a peptide as an immunizing agent against parasites wherein said peptide has the following amino acid sequence:
  • 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 Dirofilaria .
  • a vaccine for parasite infection comprising a peptide which has the following amino acid sequence:
  • a method of immunizing mammals against parasites comprising the administration of the vaccine of the present invention.
  • the vaccine of the present invention can be administered in a dosage range of 0.015 ⁇ g to 0.15 mg per kg body weight, preferably in a dosage range of 1.5 ⁇ g to 15 mg per kg body weight.
  • Fig. 1 is a Western blot analysis of extracts from parasites, pig brain and 3T3 fibroblasts using anti-B. pahangi ⁇ tubulin monoclonal antibody in Figure IA and anti-chick brain tubulin monoclonal antibody in Figure IB;
  • Fig. 2 is a Western blot analysis of the total protein extract of adult B. pahangi using anti-B. pahangi ⁇ tubulin monoclonal antibody P3D in Figure 2A or anti-B. pahangi ⁇ tubulin monoclonal antibody 1B6 in Figure 2B;
  • Fig. 3 is a Western blot analysis of products from limited proteolysis of anti-B. pahangi ⁇ tubulin monoclonal antibody P3D in Figure 3A and anti-B. pahangi ⁇ tubulin monoclonal antibody 1B6 in Figure 3B;
  • Fig. 4 is a graph of the effects of anti-B. pahangi tubulin monoclonal antibody P3D on the viability of adult female B.phangi in vitro;
  • Fig. 5 is a graph of the effects of anti-B. pahangi tubulin monoclonal antibody 1B6 on the viability of adult female B .phangi in vitro;
  • Fig. 6 is a graph of the effects of anti-chick brain tubulin monoclonal antibody 357 on the viability of adult female B.phangi in vitro. -6A-
  • 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 specifically reacts to the C-terminal portion of 3-tubulin from B . pahangi and Dirofilaria and
  • 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 of a patent to the assignee, McGill University, disclosing same. The deposit is also available as required by Foreign Patent laws in countries wherein counterpart applications are filed.
  • the monoclonal antibodies did not cross- react with tubulin from pig brain, 3T3 mouse fibroblast cells or the parasitic protozoan c muris.
  • anti-chick monoclonal antibody 357 reacted with pig brain, 3T3 mouse fibroblast and G. muris tubulins as strongly as it did with filarial and other nematode ⁇ - tubulins.
  • the anti-B. 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.
  • 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).
  • present invention are highly specific to nematode tubulin.
  • the monoclonal antibodies of the present invention specific for the - or ?-subunit of tubulin allow the subcellular localization and the function of each subunit of tubulin to be studied.
  • Proteins of the size of tubulin are generally built of several structural domains that have distinct functions. In the case of tubulin, such functions include binding of anti-microtubule drugs, GTP or microtubule-associated proteins and the association between monomers, dimers or protofilaments .
  • the nematode-specific anti-tubulin monoclonal antibodies of the present invention may serve to characterize the structure and distribution of B. pahangi tubulin molecule, and to define microtubule stability and functional domains.
  • ELISA was performed in icrotiter 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 (Guenette et al., Mol. and Biochem. Parasitology, 1991, .44.: 153- 164) at a concentration of lO ⁇ g/ml in phosphate buffer saline (PBS). Plates are incubated with 200 ⁇ l of 1% bovine serum albumin (BSA) in PBS.
  • BSA bovine serum albumin
  • Horseradish peroxidase-labeled anti-mouse IgG or IgM (Bio-Can, Mississauga, Ontario) at dilutions of 1:5000 and 1:20,000, respectively, is added to each well and incubated for 1 hour at 37°C.
  • the substrate is 2,2'- azino-bis( 3-ethylbenzthiazoline-6-sulfonic acid) (Sigma).
  • the plates are read on a Titertek multiskanTM plate (Flow Laboratories, Irvine, Ayrshire, UK) at 414 nm.
  • Samples are run in a Mini Protean II TM dual slab cell (Bio-Rad, Richmond, CA) using 4% polyacrylamide as stacking and 12% polyacrylamide as separating gels.
  • IEF gels are prepared and run in tube gels (1.5 x 8cms) containing 9.5M urea (LKB) and 2% (w/v) ampholines (LKB) (1.6% pH 4-6 and 0.4% pH 3.5-10). IEF is conducted at 400 V for a period of 16 hours and then at 800 V for 3 h. Electrophoresis is performed in 4% polyacrylamide stacking and 12% polyacrylamide separating gels, running at 50 V for 30 min and at 150 V for 60 min, in the Mini Protean II TM slab cell. After 2-dimensional (2D) SDS- PAGE, gels are either stained with silver stain (Bio- Rad), or the proteins are transferred onto nitrocellulose (NC) sheets for Western blot analysis.
  • N nitrocellulose
  • tubulin subunits After 1 . and 2D SDS-PAGE, tubulin subunits, individual tubulin isoforms and peptides are elec- trophoretically transferred onto nitrocellulose sheets for 2 hours at 4°C.
  • 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- HC1, pH 7.4, with 0.1% (v/v) Tween 20TM (TBS-T)) to saturate the unoccupied protein binding sites of the nitrocellulose.
  • TBS-T Tris-buffer saline
  • MAbs anti-tubulin monoclonal antibodies
  • 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- - - -
  • Gerbils Meriones unguiculatus ) , 9-10 months old and previously infected intraperitoneally with 400 B . pahangi infective larvae, are obtained from Dr. J. McCall (University of 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 buffer containing 1 mM ethyleneglycol-bis-( ⁇ - aminoethyl ether)N,N,N',N' -tetraacetic acid (EGTA), 0.5mM MgS ⁇ and 1 mM guanosine-5 ' -triphosphate (GTP), and are homogenized in 7 ml of 2[N-morpholino)-ethanesulfonic acid (MES) buffer. The homogenate is centrifuged at 100,000 g for 1 hour at 4°C. The supernatant is retained and the pellet discarded.
  • EGTA ethyleneglycol-bis-( ⁇ - aminoethyl ether)N,N,N',N' -tetraacetic acid
  • GTP 0.5mM MgS ⁇ and 1 mM guanosine-5 '
  • tubulin from other filarial B . malayi and D. immitis
  • non-filarial nematodes A . suum, benzimidazole- susceptible and resistant strains of H. contortus
  • Tubulin from pig brain is prepared by 2 cycles of polymerization-depolymerization.
  • Giardia Muris antigen is prepared as a sonicate.
  • KLH keyhole Limpet He ocyanin
  • Polylysine-purified proteins are separated on SDS- PAGE, protein bands of the molecular weight corresponding to tubulin are excised, and the protein is electro-eluted (ElectroeluterTM, Bio-Rad) (Blose et al. , J. of Cell Biol. , 1984, £8 . : 847-858).
  • the eluted protein is precipitated three times with 80% acetone at -20°C for 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 4°C and stored at -70°C until used.
  • Crude supernatant of adult B. pahangi is chro- matographed on a polylysine agarose column. The protein content of each fraction 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, .32 . : 145-152). Third peak proteins are concentrated and then subjected to SDS-PAGE, respectively. The tubulin band is cut out of the SDS- gels and subjected to electro-elution for further purification.
  • mice 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 . 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 of 100 ⁇ g of tubulin in PBS is administered intraperitoneally (i.p.).
  • 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.
  • 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 1B6 are raised against the tubulin of adult B . pahangi . All anti-tubulin monoclonal antibodies are of IgG isotype. 5- Specificity of monoclonal antibodies (MAbs) P3D, 1B6 and 357
  • the anti-B. pahangi monoclonal antibodies P3D and 1B6 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 microfilariae of B. pahangi , adult B malayi and D. immitis , eggs of H. contortus , adult A. suum, pig brain and 3T3 mouse fibroblast cell tubulins are separated on SDS- PAGE and electrophoretically transferred onto nitrocellulose sheets. The blots are treated with: (1) amido black; (2) monoclonal antibody 1B6; (3) monoclonal antibody P3D; and monoclonal antibody 357.
  • tubulin from the intestinal nematode H. contortus BZ-susceptible and benz- imidazole-resistant strains
  • Tubulin from A . suum do not show very strong reactivity with this monoclonal antibody
  • Fig. IA, lane 7 no reactivity to 3T3 mouse fibroblast cells or pig brain tubulins is detected (Fig. IA, lane 8-9).
  • monoclonal antibody 1B6 not shown.
  • cross-reactive anti-chick /?-tubulin monoclonal antibody 357 recognized ⁇ -tubulin from all nematodes and mammalian cells (Fig. IB, lane 1 to " 9 ) . 6- Identification of tubulin isoforms
  • Anti-B. pahangi ?-tubulin monoclonal antibodies P3D and 1B6, 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).
  • monoclonal antibody 1B6 specifically recognized only one ?-tubulin isoform in the extract of .B. pahangi (Fig. 2B).
  • the ?-tubulin isoforms are in the pH range of 5.1-5.3.
  • Monoclonal antibody 357 probed blots are re-probed with monoclonal antibodies P3D and 1B6 respectively, to demonstrate that the same spots are recognized by this monoclonal antibody. Furthermore, to show the full complement of / 0-tubulin isoforms, P3D and 1B6 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 1B6 is specific to one isoform.
  • 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 monoclonal antibodies or anti-chick tubulin monoclonal antibody 357.
  • 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: Asp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 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 :
  • the present invention provides a peptide having the amino acid sequence derived from the eighteen amino acids at the C-terminal of ⁇ -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 thereof 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.
  • a vaccine that comprises peptide with the epitope of the C-terminal of B. pahangi or Dirofilaria >3-tubulin will elicit cytotoxic antibodies in vaccinated mammals 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.
  • 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.
  • peptide of the present invention fragment thereof 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 preferred.
  • Smaller peptides according to the present invention can be synthesized, for example, by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, California) as described in detail below.
  • 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.
  • Example I in vitro assay of B. p-h-r ⁇ g ⁇ inhibition Measurement of the in vitro activity of anti-B. pahangi tubulin monoclonal antibodies P3D, 1B6 and anti- chick brain tubulin monoclonal antibody 357 against female B. 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 of MBZ drug alone or in synergy with monoclonal antibodies has any differential effect.
  • Anti-B. pahangi /?-tubulin monoclonal antibodies P3D, 1B6 (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-B. pahangi anti- chick brain monoclonal antibody 357 is in ascites fluid and is diluted to 1:1000 concentration with culture medium IMDM/FCS.
  • 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 - -
  • IMDM/FCS fetal calf serum
  • B. pahangi are washed five times with sterile IMDM/FCS medium, for surface sterilization.
  • Three wells in 24-well plates (Nunc) are set up for each test monoclonal antibody, drug and for the control cultures.
  • To each well was added 2 ml of the appropriate test medium containing pure monoclonal antibody P3D, 1B6, 357 alone or monoclonal antibody and MBZ and two adult worms.
  • the plates are incubated at 37°C in a humidified incubator in the presence of 95% air and 5% C ⁇ 2- Worm activity is observed every two hours, and motility is assessed subjectively by observation with a naked eye. Experiment is terminated after 48 hours. During the 48 hour incubation the culture medium is not changed.
  • Control medium contained an identical volume of the IMDM/FCS without monoclonal antibodies or drug.
  • Female live B. pahangi worm is placed 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 37°C for various time intervals ranging from 0-90 min (MTT-reduction) .
  • MTT 3-(4, 5-dimethyl(thiazol-2-yl)- 2,5-diphenyl tetrazolium bromide
  • 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.
  • worms are removed and carefully transferred to a separate well of a microtiter plate containing 200 ⁇ l of DMSO and allowed to stand at room temperature for 1 hour (formazan solubilization) , with occasional gentle agitation to evenly disperse the color.
  • the absorbance of the resulting formazan solution is then determined at 550 n , using an ELISA reader and compared with a DMSO blank.
  • 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- sequently diluted to 0.5 mg/ml with PBS.
  • Worms are incu ⁇ bated for 30 min at 37°C (MTT reduction). After incu ⁇ bation, worms are transferred to 96 well plates con ⁇ taining 200 ⁇ l of DMSO. The plates are allowed to stand for 1 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 100°C for 10 min.
  • 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 successful to determine filarial viability and for 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.
  • MTT assays are performed to determine the effects of anti- tubulin monoclonal antibodies on the viability of parasitic nematodes.
  • Viable control female B. pahangi showed rates of formazan formation that are maximal and linear during the first 30 min of 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-B. pahangi monoclonal antibody P3D alone and in synergy with MBZ show a detectable decrease in motility 12 hours post-treatment.
  • the other anti-B. pahangi monoclonal antibody 1B6 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 - -
  • Example I demonstrate an apparent decline in the motility, when the worms are cultured with the anti-tubulin monoclonal antibodies P3D and 1B6 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-B. pahangi , monoclonal antibodies P3D and 1B6 of the present invention, significantly reduced the viability of parasitic nematodes. No reduction in viability was observed when adult B. pahangi were exposed to anti-chick monoclonal antibody 357 and/or MBZ.
  • the carrier for such administration is an IMDM culture media.
  • the peptide consists of the amino acid sequence: sp Glu Glu Gly Asp Leu Gin Glu Gly Glu Ser Glu Tyr lie Glu Gin 1 5 10 15
  • the peptide is synthesized by solid phase methodology on an Applied Biosystems Inc. (ABI) 430A peptide synthesizer using ABI's Small Scale Rapid Cycles (SSRC) on 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 cation 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 of the peptide.
  • appropriate cation scavengers (10% v/v amisole, p-cresol plus p-thiocresol, 1,4- butanedithiol plus anisole or DMS plus anisole
  • the crude peptide after HF cleavage, is purified by preparative reverse phase chromatography on a Phenomenex C-18 Column (250 x 22.5 mm) using water acetonitrile gradients, each phase containing 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 fast atom bombardment mass spectrocopy and resulting (M+H) + is compared with the anticipated (M+H) + .
  • the peptide can be prepared in vaccine dose form by well-known procedures.
  • the vaccine can be administered sublingually, intramuscularly, subcuta- neously or intranasally.
  • 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 dipeptide, bacterial endotoxin, lipid X, Corynebacterium parum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium - 24 -
  • alginate alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Co., Inc., Rahway, NJ) .
  • the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (AI2O3 basis).
  • the concentration of the immunogen can range from about 0.015 ⁇ g to about 1.5 mg per kilogram per patient body weight for an animal or human patient.
  • a preferable dosage range in humans is about 0.1 - 1 ml, preferably about 0.1 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.1 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP93922866A 1992-10-28 1993-10-22 Nematodentubulin abgeleitete peptide und impfstoffe Withdrawn EP0674659A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US967829 1978-12-08
US96782992A 1992-10-28 1992-10-28
PCT/CA1993/000431 WO1994010201A1 (en) 1992-10-28 1993-10-22 Peptides and vaccines derived from nematode tubulin

Publications (1)

Publication Number Publication Date
EP0674659A1 true EP0674659A1 (de) 1995-10-04

Family

ID=25513395

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93922866A Withdrawn EP0674659A1 (de) 1992-10-28 1993-10-22 Nematodentubulin abgeleitete peptide und impfstoffe

Country Status (6)

Country Link
EP (1) EP0674659A1 (de)
JP (1) JPH08503935A (de)
KR (1) KR950704359A (de)
AU (1) AU5282193A (de)
CA (1) CA2147756A1 (de)
WO (1) WO1994010201A1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2404270A (en) 2003-07-24 2005-01-26 Hewlett Packard Development Co Document composition

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0093775A1 (de) * 1981-11-17 1983-11-16 Brigham And Women's Hospital Monoklonale antikörper gegen brugia malayi
GB2152510B (en) * 1984-01-10 1988-06-15 Kenneth K Lew Method for the commercial production of helminths antigens
NL9001832A (nl) * 1990-08-16 1992-03-16 Rijksuniversiteit Specifieke sequenties van dna van een nematode die toegepast kunnen worden bij de diagnostiek van infektie met de nematode.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9410201A1 *

Also Published As

Publication number Publication date
WO1994010201A1 (en) 1994-05-11
JPH08503935A (ja) 1996-04-30
CA2147756A1 (en) 1994-05-11
AU5282193A (en) 1994-05-24
KR950704359A (ko) 1995-11-20

Similar Documents

Publication Publication Date Title
CA1341166C (en) Antigenic proteins and vaccines containing them for prevention of coccidiosis
Newton Progress on vaccination against Haemonchus contortus
WO1997036612A1 (en) Anti-cryptosporidium parvum preparations
US6015882A (en) Vaccines, antibodies, proteins, glycoproteins, DNAs and RNAs for prophylaxis and treatment of Cryptosporidium parvum infections
KR100244828B1 (ko) 콕시디아증 백신
KR100553646B1 (ko) 말라리아원충 항원 폴리펩티드 에스이36, 그 정제방법,그리고 이것으로부터 얻어지는 항원을 이용하는 백신 및진단제
KR20010011330A (ko) 한국산 겨우살이 추출물과 이로부터 분리한 단백질 및 상기 단백질에서 분리한 렉틴
AU759316B2 (en) Chimeric gene encoding the antigenic determinants of four proteins of L. infantum
WO1997006263A1 (en) Hybrid protein comprising t-helper cell stimulating epitopes and b-cell epitopes from the major outer membrane protein of chlamydia trachomatis and its use as a vaccine
AU2017261603B2 (en) Tick toxin compositions
AU663862B2 (en) Nematode vaccine
WO1994010201A1 (en) Peptides and vaccines derived from nematode tubulin
FR2863499A1 (fr) Nouveaux vaccins destines au traitement ou a la prevention des infections par parasites de la famille echinococcus
US20180311327A1 (en) Aquaporin 2 protects cattle from ticks and tick-borne parasites
JP3582663B2 (ja) 鶏ロイコチトゾーン免疫原性蛋白質を発現する遺伝子クローン及び鶏ロイコチトゾーン症に対する遺伝子組換えワクチン
EP0283829A1 (de) Plasmodium falciparum Merozoitenantigen-Peptide
WO1996032132A1 (en) Peptides of nematode tubulin and methods of use
US6962704B2 (en) Chimeric gene formed by the dna sequences that encode the antigenic determinants of four proteins of l. infantum and protein encoded by said gene, and pharmaceutical composition useful for preventing and/or treating leishmaniosis in animals or humans
Sawangjaroen et al. Immunohistochemical localization of excretory/secretory antigens in adult Ancylostoma caninum using monoclonal antibodies and infected human sera
CA2256124C (en) Chimeric gene formed of the dna sequences that encode the antigenic determinants of four proteins of l. infantum, and protein encoded by said gene, and pharmaceutical composition useful for preventing and/or treating leishmaniosis in animals or humans
NZ224597A (en) Taenia ovis antigens and vaccines
FR2850384A1 (fr) Antigenes mimant les domaines extracellulaires de proteines membranaires de type iii issues de microorganismes intracellulaires pathogenes, anticorps conformationnels derives, et leurs applications
GB2219590A (en) Antigenic polypeptides of taena ovis
CA2253834A1 (en) B. burgdorferi polypeptides expressed in vivo
WO2006009862A2 (en) Amebiasis subunit vaccine

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19950529

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19980501